Targeted gastrointestinal tract delivery of probiotic organisms and/or therapeutic agents

ABSTRACT

The present invention restores altered probiotic organism imbalances that are characteristic of said diseases among others as well as defines a platform technology development for site specific delivery of probiotic organisms in the GI tract of a mammal, most specifically the ileum and/or right colon of a human subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/771,830, filed on Sep. 1, 2015, which is filed under the provisionsof 35 U.S.C § 371 and claims the priority of International PatentApplication No. PCT/US2014/027228 filed on Mar. 14, 2014, which in turnclaims priority to U.S. Provisional Application No. 61/781,810 filed onMar. 14, 2013, and U.S. Provisional Application No. 61/897,378 filed onOct. 30, 2013, the contents of which are incorporated by referenceherein for all purposes.

FIELD OF THE INVENTION

The present invention relates to the development of platform technologyfor targeted, controlled delivery of oral enhanced probiotics forvarious indications, including for example the active and prophylaxistreatment of Clostridium difficile Infection as well as Metabolicsyndrome and type 2 diabetes.

BACKGROUND OF THE INVENTION

The following includes information that may be useful in understandingthe present inventions. It is not an admission that any of theinformation provided herein is prior art, or relevant, to the presentlydescribed or claimed inventions, or that any publication or documentthat is specifically or implicitly referenced is prior art.

Recent studies have highlighted the importance of the human microbiomein health and disease. However, for the most part the mechanisms bywhich the microbiome mediates disease, or protection from it, remainpoorly understood. Hajishengallis and colleagues have been developingthe Keystone-pathogen hypothesis, which highlights the importantinteractions between flora normally found in healthy humans, diseasesassociated with alterations in these flora, and the role of the hostinflammatory system in the transition between health and a disease state(1). The keystone-pathogen hypothesis holds that certain low-abundancemicrobial pathogens can orchestrate inflammatory diseases, by remodelinga normally benign microbiota into a dysbiotic one. Hajishengallis andcolleagues critically assess the available literature that supports thekeystone hypothesis, which may provide a novel conceptual basis for thedevelopment of targeted diagnostics and treatments for complex dysbioticdiseases. This work provides an elementary background understanding foruse of specific organisms delivered to specific sites in theGastro-intestinal tract, which is the subject of the instant invention.

As currently understood, probiotics are live non-toxic microbial foodsupplements that can beneficially affect a host by improving the host'sintestinal microbial balance without causing disease. Because probioticorganisms may be altered by antibiotic treatments or for other reasons,they do not permanently colonize in the body. It is therefore importantthat they be ingested regularly for their health-promoting effects topersist. After ingestion, probiotics typically adhere to a tissue of thehost, such as the wall of the intestine or the vagina. Once attached,the desirable bacteria are capable of multiplying and colonizing,thereby enhancing optimal microflora balance. They are used to promotehealthy microflora (‘good bacteria” or commensals) balance (good oreubiosis) in the lower GI tract and healthy pH balance (yeast fungus) inthe oral cavity, large intestine and vaginal tract and minimizemicrobial imbalance or dysbiosis. Probiotics characteristics are thefollowing: (1) from human origin; (2) stable and viable, gastric andbile acid resistant; (3) effectively adhere to and colonizing at thesite of action; (4) compete with pathogens for adhesion sites; and (5)produce pathogen inhibitory substances, e.g. bacteriocidins and organicacids.

Probiotics provide: (1) normalization of flora (e.g., suppress PPMs,provide for intestinal mucosal integrity, regulation of bowel movement,IBS, etc.); (2) Immunomodulation (e.g., strengthen immunity, alleviatefood allergy symptoms, control of IBD, etc.); (3) Metabolic effects(e.g., Production of vitamins to improve digestion, minimize lactoseintolerance, lower cholesterol, promote bile acid deconjugates, etc.)and many other benefits. Probiotics are sometimes combined withprebiotics (combination is called Symbiotic) which are range of range ofnon-digestible dietary supplements, which modify the balance of theintestinal micro flora, stimulating the growth and/or activity ofbeneficial microorganisms and suppressing potentially deleteriousmicroorganisms. The supplements include oligosaccharides(fructo-oligosaccharides, galacto-oligosaccharides); Inulin, Lactulose,Lactitol and a few select bacterial strains that produce bifidogenicnutrients. In particular, prebiotics promote the proliferation ofBifidobacteria in the colon and also promote the proliferation ofLactobacilli in the small intestine to a certain extent.

There are many nutritional probiotics products currently available andare marketed as dietary supplements with very soft DSHEA type “supporthealth” benefit claims. Probiotic products are marketed in all differenttypes of dosage forms, by way of example liquids, capsules, entericcoated tablets and matrix sustained release formulations for oraladministration. They use different mix of bacteria and sometimes areenteric coated and of the type which are conventionally released intoduodenal target and would not survive transit to reach the potentialtarget organs, e.g., colon. The normal pH profile of the GI tractchanges (up and down) from the stomach to the colon, e.g. the pH of thestomach, duodenum, ileum and colon is in the range of 1-4, 5.5-6,7.3-8.0 and 5.5-6.5, respectively. In some diseases conditions the pH ofthe GI tract may be modified, e.g. pH of the ileum in normal is 7.5 to8.2, while pH of the ileum in Metabolic Syndrome, Type 2 diabetes andObese subjects is 7.3 to 7.5, as discovered using the SmartPill toexamine distal intestinal pH values in health and diseases.

To date it is assumed that there are no published reports of any kindthat support any specific US FDA approved clinical efficacy or safetyclaims, nor delivery to any specific area or specific benefits of theprobiotics. All of the current evidence is generated from differentsystems and has not been utilized for a practical treatment regimen thatis directed toward flora replacement strategy prior to our foundationdiscoveries in Roux-en-Y gastric bypass (RYGB) patients (3) Likewise, noproduct currently exists that specifically delivers the probioticorganism(s) at the target specific pH of the colon at pH 5.5-6.2. Mostof the enteric products release the probiotic to the duodenum at pH5.5-6.2 and because of degradation in the proximal intestine, organismsreleased may never actually reach the ileum or the right sided colon.Accordingly, it would be advantageous to develop a platform technologyfor controlled delivery formulation of oral enhanced probiotics thatspecifically target to release in the pH environment of the ileum andthe colon, for treatment/cure of various diseases (pill in a pillconcept). These include the active and prophylaxis treatment forClostridium difficile infection, and possible treatments of metabolicsyndrome in diabetes.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides one or more species ofmicroencapsulated live probiotic organisms that have a biphasic releaseprofile in a subject. The one or more species of microencapsulated liveprobiotic organisms provided herein may be in the form of a formulation(e.g. in the form of a tablet, capsule, or the like), wherein theformulation comprises one, or more than one species of bacteria that arenormally present in the intestine of a subject.

In certain preferred embodiments, this biphasic release profile has arelease profile in a subject such that living organisms are firstreleased in a subject at pH values between about 7.0 and 8.0, andsecondly to the first release, living organisms are subsequentlyreleased at pH values of between about 5.5 and 6.0.

In another aspect, the invention provides microencapsulated liveprobiotic organisms that have a release profile that targets replacementor revision of one or more species of live bacteria at a pre-determinedlocation within the gastrointestinal tract of a mammal. As will bedescribed in greater detail herein below certain embodiments areprovided wherein the pre-determined location within the gastrointestinaltract is the ileum or colon and other embodiments wherein theformulations provided have a pH dependent preferential release and sitespecific release of a probiotic organism in the intestinal tract of amammal. Said organism replacements may be made specifically to modifythe course of metabolic syndrome associated diseases such as obesity,type 2 diabetes, or the like. Said organism replacements may also bemade in other preferred embodiments of the invention to repairintestinal dysbiosis associated diseases, such as, Antibiotic associateddiarrhea (AAD), Clostridium difficile associated diarrhea (CDAD),metabolic syndrome, etc. Each of these conditions will require specificmicrobiome replacements or restorations as will be disclosed herein.

In one preferred embodiment provided herein, the microencapsulated liveprobiotic organisms having a release profile in which one or morespecies of live probiotic organisms is released in the into the ileum ofa subject in an area having a pH of from about 7 to 8.

In another aspect, the probiotic organism provided in certainembodiments is a mixture of bacterial genera in the amounts that arereflective of the mixture of strains derived from the ileum of a normalhuman, in amounts that replace these genera reflective of normalintestinal balance. Typically, the number of said organisms released ismore than 10⁵ and less than 10¹², where the probiotic organism is amixture of bacterial genera that is reflective of the mixture of strainsderived from the stool of a normal human, but it is appreciated thatthese numbers are not limiting and that lower of higher amounts of anylive organism that is administered may be lower or higher than theseamounts.

In another aspect, compositions and methods are provided to amelioratethe imbalance of Clostridium difficile in a subject suffering from suchan imbalance. Accordingly, in certain embodiments, one or more speciesof microencapsulated live probiotic organisms having a biphasic releaseprofile results in a release of these live probiotic organisms into thedistal segments of the gastrointestinal tract, including the ileum andcolon of a subject, in order to ameliorate the imbalance of Clostridiumdifficile in a subject suffering from such an imbalance.

In another aspect, formulations are provided herein for the protectionof the live probiotic organisms from the digestive actions of thestomach, duodenum, and jejunum of the intestine. Accordingly, someembodiments provide one or more species of microencapsulated liveprobiotic organisms as a formulation that provides protection of thelive probiotic organisms from the digestive actions of the stomach,duodenum, and jejunum of the intestine, such that the desired number oforganisms is administered to the ileum of a subject.

In some embodiments, formulations provided herein comprise anencapsulated live probiotic from which one or more probiotic bacteriaare dispersed, the encapsulated probiotic comprising a coatingcomprising “polymers”. In certain embodiments, a live bacterialsuspension including species from one or both of the genus'sLactobacillus and Bifidobacterium is provided. In alternativeembodiments, live bacterial suspensions including species from one orboth of the genus's Lactobacillus and Bifidobacterium and furthercomprising the organism Faecalibacterium prausnitzii are provided. Inyet another alternative embodiment, live bacterial suspensions includingspecies from one or both of the genus's Lactobacillus andBifidobacterium and further comprising the organism Bacteroidesthetaiotaomicron are provided.

In yet another aspect, one or more species of microencapsulated liveprobiotic organisms are provided in which the microencapsulated liveprobiotic organisms have a three phase release profile. Accordingly, ina fundamental embodiment of this aspect of the invention, one or morespecies of microencapsulated live probiotic organisms are providedwherein the microencapsulated live probiotic organisms have a threephase release profile in a subject in which living organisms arereleased in a subject i) at pH values between about 5.5-6.2 such thatthe live probiotic organisms are released in the duodenum, ii) at pHvalues between about 7.2-7.5 such that the live probiotic organisms arereleased in the ileum, and iii) at pH values between about 5.6-6.2 suchthat the live probiotic organisms are released in the colon.

In certain preferred variations of embodiments provided above, it isfurther desirable that none of the bacterial organisms are released inthe small intestine at pH values below 6.9 or above 8.1. Thus, the areaof release will be within the intestinal tract that includes a highlevel of Peyer's Patches, that being the ileum.

In another variation of the fundamental embodiment provided above, therei) is an outer layer of microencapsulated probiotic organisms withrelease characteristics between pH values of 7.0 to 8.0, and ii) aprotected inner core of microencapsulated probiotic organisms that arereleased at pH values below pH of 6.9. This will allow the probiotic tobe released in the ileum and colon of the subject.

In certain embodiments, one or more species of microencapsulated liveprobiotic organisms are provided where the organism specificallystimulates L-cell expression of proteins, hormones or biomarkers ofL-cell actions therefrom. In additional embodiments, one or more speciesof microencapsulated live probiotic organisms are provided wherein theprobiotic organisms specifically metabolize bile acids in the distalintestine of the mammal, and where the formulation has beneficialactions on cholesterol and triglyceride concentrations in a mammal.

In still another aspect of the invention, methods of treatment of asubject are provided (e.g. a mammal or human). Accordingly, in certainembodiments a method of treating a Clostridium difficile associatedintestinal disorder in a subject is provided in which said methodcomprises administering a formulation claimed or otherwise providedherein in an amount sufficient to alleviate the disorder being treatedin a subject. A clostridium difficile associated disorder treated by theformulation and methods provided herein may be associated with one ormore of a Clostridium difficile infection, an imbalance of Clostridiumdifficile in the ileum or colon of said subject, diarrhea, inflammation,colitis fever, or the like. Administration of the formulations bymethods provided herein alleviates one or more of the preceding signsand symptoms of infection with Clostridium difficile. It is preferablethat such treatment results in the prophylaxis or prevention of aClostridium difficile infection.

In another aspect, kits comprising one or more species of encapsulatedmicroorganisms and formulations of the same are provided herein.Accordingly, some embodiments of the invention are directed to a kitcomprising encapsulated microorganisms and formulations claimed orotherwise provided herein in the form of a tablet, pill, capsule orsachet of microgranules in combination with instructions foradministration of the formulation to a subject for the treatment of adisorder. Certain preferred embodiments of the kit are designed for thetreatment of a Clostridium difficile associated disorder in a subjectsuffering from such a disorder.

In yet another aspect, kits containing one or more species ofencapsulated microorganisms and formulations of same are provided withinstructions to patients in need of the procedure termed “fecaltransplant” wherein the microgranules of the present invention andformulations are provided herein in the form of a tablet, pill, orcapsule in combination with instructions for administration of theformulation to a subject in need of a fecal transplant. Certainpreferred embodiments of said kit are designed for the treatment of aClostridium difficile associated disorder in a subject suffering fromsuch a disorder.

Microencapsulated live probiotic organisms and formulations thereof areprovided herein in various dosage forms, and they can be co-administeredwith drugs, foods, nutrients, vitamins, other beneficial substances,prebiotics, and other therapeutic agents such as pH encapsulatedglucose, lipids or proteins that release in the distal small intestineat pH values between 7.0 and 8.0 in an amount sufficient to alleviatesaid disorder in a subject. Preferably, at least two coating are used tocover a tablet or capsule like form comprising the probiotic organism,wherein the outside coating is degraded in a pH environment of 5 to 6and the inside coating is degraded in a pH environment of about 7thereby dropping the probiotics in the ileum area and in close proximityto the Peyer's Patches.

In certain embodiments, microencapsulated live probiotic organisms andformulations thereof are administered in conjunction with one or moreantibiotic. The dosage formulation is designed in these embodiments tocompletely separate the antibiotic from the bacteria, and testing isconducted to verify complete separation on a long term basis. Suitableantibiotics include, but are not limited to, vancomycin, metronidazole,gentamicin, colistin, fidaxomicin, telavancin, oritavancin, dalbavancin,daptomycin. An exemplary embodiment is directed to one or more speciesof microencapsulated live probiotic organisms claimed or otherwiseprovided herein in a dosage of between 10⁵ and 10¹² CFU, wherein thedosage unit of the formulation contains vancomycin at a dose of betweenabout 125 mg to about 4000 mg, wherein the antibiotics released fromeach dosage unit formulation at between about pH 1.0 to about pH 6.0. Incertain embodiments, microencapsulated live probiotic organisms andformulations thereof are co-administered with vancomycin in an effectiveamount for the beneficial treatment of Clostridium difficile infectionor complications thereof.

In still another aspect, the microencapsulated live probiotic organismsclaimed or otherwise provided herein are used for the treatment of otherdisorders. In non-limiting but preferred embodiments described herein,antibiotics are not included in the formulation.

One embodiment is directed to a method of treating an obesity-associatedintestinal disorder in a subject, where the method comprisesadministering a probiotic formulation targeted to the ileum and rightcolon which is claimed or otherwise provided herein an amount sufficientto alleviate the disorder in said subject. Another embodiment isdirected to a method of treating type 2 diabetes associated metabolicsyndrome, where the method comprises administering a probioticformulation targeted to the ileum and right colon which is claimed orotherwise provided herein an amount sufficient to alleviate the disorderin said subject. In a variation of this embodiment, the organism(s)being used are capable of signaling the release of GLP-1, PYY, GLP-2 orother beneficial peptides from the L-cell target site in the intestine,whereby the disease or condition or metabolic syndrome is modifiedbeneficially. An example of this modification is the treatment of type 2diabetes with said probiotic formulation in combination with an ilealbrake hormone releasing substance active at the ileal brake, where bothactive moieties act to stimulate L-cell hormone release and to revisesignaling of hormones. Replacement of numbers and specific species ofprobiotic organisms in targeted ileum and colon produces homeostatic andbeneficial regulation of L-cell hormone release from the ileum and rightsided colon. These novel approaches to treatment are disclosed herein inspecific examples.

Another preferred embodiment includes treatment with an anti-diabeticdrug, an ileal brake hormone releasing substance and a probioticorganism, wherein said probiotic organism replacement or revision isdirected to one or more species of microencapsulated live probioticorganisms claimed or otherwise provided herein in a dosage of between10⁵ and 10¹² CFU, wherein the dosage unit of the formulation containsmetformin at a dose of between about 250 mg to about 1000 mg, whereinthe metformin released is from each dosage unit formulation at betweenabout pH 1.0 to about pH 6.0. In certain embodiments, microencapsulatedlive probiotic organisms and formulations thereof are co-administeredwith metformin in an effective amount and are co-administered with about5.0 gm to about 10.0 grams of microgranules of dextrose and nutritionalsubstances, as disclosed in US20110268795, said formulation encapsulatedfor release at intestinal pH between 7.0 and 7.5, said combinationdisclosed herein known to be beneficial in the treatment of Type 2diabetes, metabolic syndromes or complications thereof.

Another embodiment is directed to one or more species ofmicroencapsulated live probiotic organisms claimed or otherwise providedherein in a dosage of between 10⁵ and 10¹² CFU, wherein the dosage unitof the formulation contains atorvastatin at a dose of between about 10mg to about 80 mg, wherein the atorvastatin released is from each dosageunit formulation at between about pH 1.0 to about pH 6.0. In certainembodiments, microencapsulated live probiotic organisms and formulationsthereof are co-administered with atorvastatin in an effective amount andare co-administered with about 5.0 grams to about 10.0 grams ofmicrogranules of dextrose and nutritional substances, as disclosed inUS20110268795, said formulation encapsulated for release at intestinalpH between 7.0 and 7.5, said combination disclosed herein known to bebeneficial in the treatment of Type 2 diabetes, hyperlipidemia,metabolic syndrome or complications thereof.

In certain preferred embodiments, microencapsulated live probioticorganisms and formulations thereof are co-administered with TumorNecrosis Factor (TNF) antagonist in an effective amount encapsulated forrelease at intestinal pH between 7.0 and 7.5, said combination disclosedherein known to be beneficial in the treatment of Crohn's disease,Ulcerative colitis, inflammatory bowel disease or the like, orcomplications thereof.

Another embodiment is directed to a method of treating irritable boweldiseases associated with dysbiosis, where the method comprisesadministering a probiotic formulation targeted to the ileum and rightcolon which is claimed or otherwise provided herein an amount sufficientto alleviate the disorder in said subject. In certain embodiments themicroencapsulated live probiotic organisms and formulations thereof areco-administered with drug treatments approved for treatment of irritablebowel diseases, such as linaclotide. Non-limiting examples of irritablebowel diseases and treatments thereof are contained within theseembodiments.

Yet another aspect of the present invention is an oral delivery systemthat delivers a probiotic formulation targeted to the ileum and rightcolon of a subject; the system comprising:

-   -   a core comprising a probiotic formulation; and    -   a coating which encapsulates the probiotic formulation, which is        substantially insoluble at a pH of less than a range of between        about 7.0 to about 8.0 and soluble in the pH range of about 7.0        to about 8.0, and wherein the probiotic formulation is not        released until the pH is about 7 and there is essentially no        loss of the probiotic formulation through the digestive tract        until the delivery systems reaches the ileum.

Preferably, the coating is comprised of one or more compositionsselected from the group consisting of poly(dl-lactide-co-glycolide,chitosan (Chi) stabilized with PVA (poly-vinylic alcohol), a lipid, analginate, carboxymethylethylcellulose (CMEC), cellulose acetatetrimellitiate (CAT), hydroxypropylmethyl cellulose phthalate (HPMCP),hydroxypropylmethyl cellulose, ethyl cellulose, color con, food glazeand mixtures of hydroxypropylmethyl cellulose and ethyl cellulose,polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP),shellac, copolymers of methacrylic acid and ethyl acrylate, andcopolymers of methacrylic acid and ethyl acrylate to which a monomer ofmethylacrylate has been added during polymerization,

In yet another aspect, the present invention provides for an oraldelivery system for delivering a probiotic formulation targeted to theileum and proximal colon of a subject; the system comprising:

-   -   a core comprising a probiotic formulation wherein the probiotic        formulation is included in a biodegradable first capsule that is        coated with a first enteric coating that encapsulates the first        capsule containing the probiotic formulation, and wherein the        first enteric coating solubilizes in a pH of about 6.2 to about        6.5; and    -   a second capsule sized to include the coated first capsule,        wherein the second capsule is fabricated of a biodegradable        material and wherein the second capsule is coated with a second        enteric coating that solubilizes in a pH of about 7 to 8,        wherein the second capsule releases the first capsule in the        ileum and once released the first capsule is solubilized in the        proximal colon at a pH of about 6.2 to about 6.5 with the        release of the desirable bacteria.

Importantly, the second enteric coating is substantially insoluble at apH of less than a range of between about 7.0 to about 8.0 and soluble inthe pH range of about 7.0 to about 8.0. The first and second entericcoatings are comprised of one or more compositions selected from thegroup consisting of copolymers of methacrylic acid and ethyl acrylate,and copolymers of methacrylic acid and ethyl acrylate to which a monomerof methylacrylate has been added during polymerization. Notably, thesecond capsule releases the first capsule in the ileum and once releasedthe first capsule is solubilized in the proximal colon at a pH of about6.2 to about 6.5 with the release of the probiotic formulation.

The probiotic formulation comprises at least one species of bacteria,preferably from 1 to 30, and more preferably from about 10 to 25different species or strains, that are normally present in apre-determined location within the gastrointestinal tract of a subjectand preferably the pre-determined location is the ileum or colon. Thespecies of bacteria may be different or just include different strains.The probiotic formulation comprises a mixture of bacterial genera thatis reflective of the mixture of strains derived from the ileum of anormal human, and the number of said organisms released is more than 10⁶and less than 10¹². Preferably, the release of the probiotic formulationis in the distal segments of the gastrointestinal tract including theileum and colon of a subject and to ameliorate the imbalance ofClostridium difficile in a subject suffering from such an imbalance. Aneffective probiotic formulation comprises a live bacterial suspensionselected from the genus Lactobacillus and Bifidobacterium. Such aformulation may further comprise the organism Faecalibacteriumprausnitzii.

The probiotic formulation can be combined with drugs, acetaminophen,foods, nutrients, vitamins, beneficial substances, prebiotics, pHencapsulated glucose, lipids or proteins that release in combinationwith the probiotics or in a pH of from about 1 to 6 and before therelease of the probiotics. Also the probiotic formulation may also beco-administered with an antibiotic selected from the group consisting ofvancomycin, metronidazole, gentamicin, colistin, fidaxomicin,telavancin, oritavancin, dalbavancin and daptomycin. Still further theprobiotic formulation may be combined with an ileal brake hormonereleasing substance active at the ileal brake to stimulate L-cellhormone release and to revise signaling of hormones.

The probiotic formulation may be used to modify the course of metabolicsyndrome associated diseases selected from the group consisting ofobesity and type 2 diabetes; or to repair intestinal dysbiosisassociated diseases selected from the group consisting of Antibioticassociated diarrhea (AAD), Clostridium difficile associated diarrhea(CDAD) and metabolic syndrome.

The probiotic formulation may also be combined with an anti-diabeticdrug, such as metformin; a statin, such as atorvastatin; or ananti-inflammatory, such as a Tumor Necrosis Factor (TNF) antagonist.

Another aspect of the present invention provides for acapsule-in-capsule oral delivery system that delivers desirableprobiotics or therapeutic agents to the ileum and/or proximal colon, thesystem comprising:

-   -   a first capsule containing the desirable probiotics or        therapeutic agents, wherein the first capsule is fabricated of a        biodegradable material and wherein the first capsule is coated        with a first enteric coating that solubilizes in a pH of about        6.2 to about 6.5; and    -   a second capsule being of a size that can include within its        dimensions the coated first capsule, wherein the second capsule        is fabricated of a biodegradable material and wherein the second        capsule is coated with a second enteric coating that solubilizes        in a pH of about 7 to 8, wherein the second capsule releases the        first capsule in the ileum and once released the first capsule        is solubilized in the proximal colon at a pH of about 6.2 to        about 6.5 with the release of the desirable probiotics or        therapeutic agents.

Notably, the second capsule may further comprise desirable probioticsfor release in the ileum. Importantly, the desirable probiotics ortherapeutic agents within the capsule system are delivered to the ileumand/or proximal colon without leakage of such probiotics or therapeuticagents in the proximal areas of the gastrointestinal tract positionedbefore the ileum and/or proximal colon. The present system provides forat least 90% of the desirable probiotics or therapeutic agents to reachthe ileum and/or right colon, more preferably at least 95%, and mostpreferably at least 97%.

The first and second enteric coatings of the capsule-in-capsule oraldelivery system are preferably selected from the group consisting ofcopolymers of methacrylic acid and ethyl acrylate, and copolymers ofmethacrylic acid, methyl acrylate and methyl methacrylate.

The capsule-in-capsule oral delivery system provide for a system whereinthe wherein the outside and firstly exposed second capsule releases thefirst capsule in the ileum and once released the first capsule issolubilized in the proximal colon at a pH of about 6.2 to about 6.5 withthe release of the desirable probiotics or therapeutic agents. If thecontent is desirable probiotics then such probiotics comprise at leastone to 30 species of bacteria, more preferably from about 10 to 25different species or strains of such species that are normally presentin a pre-determined location within the gastrointestinal tract of asubject and preferable the pre-determined location is the ileum orcolon. The desirable probiotics may comprise a mixture of bacterialgenera that is reflective of the mixture of strains derived from theileum of a normal human, and the number of said organisms released ismore than 10⁵ and less than 10¹² and preferably the release is in thedistal segments of the gastrointestinal tract including the ileum andcolon of a subject and to ameliorate the imbalance of Clostridiumdifficile in a subject suffering from such an imbalance. Such probioticscomprise a live bacterial suspension selected from the genusLactobacillus and Bifidobacterium and may further the organismFaecalibacterium prausnitzii.

A very effective combination of coating for the capsule-in-capsule oraldelivery system comprises a the first capsule is coated (first entericcoating) with about 10 mg/cm² of EUDRAGIT® EPO and the second capsule iscoated (second enteric coating) with about 5 mg/cm² of EUDRAGIT®L100/S100, 75/25 mix wherein the capsules are fabricated fromhydroxypropylmethyl cellulose.

Still another aspect of the present invention provides for a method oftreating the onset of a gastrointestinal disorder, the method comprisingadministering to a subject in need of such treatment in apharmaceutically effective amount of an oral formulation comprising:

-   -   a first capsule containing desirable probiotics having        beneficial effects on a gastrointestinal disorder, wherein the        first capsule is fabricated of a biodegradable material and        wherein the first capsule is coated with a first enteric coating        that solubilizes in a pH of about 6.2 to about 6.5; and    -   a second capsule being of a size that can include within its        dimensions the coated first capsule, wherein the second capsule        is fabricated of a biodegradable material and wherein the second        capsule is coated with a second enteric coating that solubilizes        in a pH of about 7 to 8, wherein the second capsule releases the        first capsule in the ileum and once released the first capsule        is solubilized in the proximal colon at a pH of about 6.2 to        about 6.5 with the release of the desirable probiotics.

The gastrointestinal disorder includes a Clostridium difficile disorderthat is associated with one or more of a Clostridium difficileinfection, an imbalance of Clostridium difficile in the ileum or colonof said subject, diarrhea, inflammation, colitis fever, and wherein theoral formulation is in an amount sufficient to alleviate thegastrointestinal disorder in the subject and comprises a live bacterialsuspension selected from the genus Lactobacillus and Bifidobacterium.

In a still further aspect, the present invention provides for the use ofan oral formulation for preparing a medicament for the treatment ofgastrointestinal disorder wherein the oral formulation comprises:

-   -   a first capsule containing a desirable bacteria effective        against the gastrointestinal disorder, wherein the first capsule        is fabricated of a biodegradable material and wherein the first        capsule is coated with an enteric coating that solubilizes in a        pH of about 6.2 to about 6.5; and    -   a second capsule being of a size that can include within its        dimensions the coated first capsule, wherein the second capsule        is fabricated of a biodegradable material and wherein the second        capsule is coated with an enteric coating that solubilizes in a        pH of about 7 to 8, wherein the second capsule releases the        first capsule in the ileum and once released the first capsule        is solubilized in the proximal colon at a pH of about 6.2 to        about 6.5 with the release of the desirable bacteria.

Another aspect of the present invention provides for an oral deliverysystem to deliver an oral formulation targeted directly to the ileumand/or colon of a subject with essentially no loss of the oralformulation before reaching at least the ileum, the system comprising:

-   -   a core comprising the oral formulation, wherein the oral        formulation comprises probiotics or a therapeutic agent;    -   a first enteric coating encapsulating the core, wherein the        first coating dissolves in a dissolution pH of about 6.2 to        about 6.5;    -   a second enteric coating encapsulating the first coating,        wherein the second coating dissolves in a dissolution pH of        about 7 to 8.

Preferably, this oral delivery system further comprising a firstbiodegradable film layer positioned between the core and first coatingand also a second biodegradable film layer positioned between the firstcoating and the second coating, wherein the biodegradable film ishydroxypropylmethyl cellulose.

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the Distal Intestine Regulatory component of MetaSensor andassociated host Metabolomics-Interactions between L-cells and Probioticbacteria.

FIG. 2 shows normal operations of the MetaSensor via stop signals GLP-1,PYY and other L-cell derived regulatory hormones.

FIG. 3 shows the situation when a diabetogenic food, such as sugarsweetened beverage alters the microbiome and thus the hormonal operationof the MetaSensor.

FIG. 4 shows the situation when there is a Microbiome dysbiosis thatproduces abnormal regulatory control of the MetaSensor via its action onthe L-cells.

FIG. 5 shows the impact of Roux-en-Y gastric bypass (RYGB) surgery onthe MetaSensor.

FIG. 6 shows the impact of an oral mimetic of RYGB, an ileal brakehormone releasing substance called Brake, on the MetaSensor.

FIG. 7 considers the impact of a common diabetes drug, metformin, incombined with Brake, on the operations of the MetaSensory process,illustrating synergistic interactions between a drug and a mimetic ofRYGB surgery.

FIG. 8 shows the dissolution of Acetaminophen (APAP) 325 mg core tabletsin pH 6.5 (USP dissolution apparatus: Basket at 50 rpm; n=3).

FIG. 9 shows the dissolution profile of 325 mg APAP tablets sealed with4 mg/cm² seal (HPMC) and coated with EUDRAGIT®-EPO 18 cm²) in pH 6.0, pH6.5, pH 6.8, pH 7.0 and pH 7.4. (USP dissolution apparatus: Basket at 50rpm; n=3).

FIG. 10 shows the dissolution of 325 mg APAP core tablet in pH 7.0 (USPdissolution apparatus: Basket at 50 rpm; n=3).

FIG. 11 shows the dissolution profile of 325 mg coated APAP tabletscoated with different ratios of FS30 D & L30D55 in pH 1.2, pH 5.5 & pH7.0. (USP dissolution apparatus: Basket at 50 rpm; n=3).

FIG. 12 shows the comparison of dissolution profiles of uncoated APAP(˜91 mg) capsules (size #3) in pH 6.5 (using two USP dissolutionapparatus: basket @ 75 rpm and Paddle @ 50 rpm; n=3).

FIG. 13 shows the comparison of dissolution profiles of coated APAP (˜91mg) capsules (size #3) coated with 10 mg/cm² EUDRAGIT®-EPO in pH 6.5(using two USP dissolution apparatus: basket @ 75 rpm and Paddle @ 50rpm; n=3).

FIG. 14 shows the dissolution profile of coated APAP (˜91 mg) capsules(size #3) coated with EUDRAGIT®-EPO (10 mg/cm²) in pH 6.8 (USPdissolution apparatus: Paddle at 100 rpm; n=3).

FIG. 15 shows the dissolution profile of uncoated APAP (˜335 mg)capsules (size #0) sealed with 6 mg/cm² HPMC in pH 6.5 (USP dissolutionapparatus: Paddle at 100 rpm; n=3).

FIG. 16 shows the dissolution profile of APAP (˜335 mg) capsules (size#0), sealed with HPMC-6 mg/cm2 and coated with EUDRAGIT® L100 &EUDRAGIT®-L100/S100 (50/50)—7.5 mg/cm² in multi-media (USP dissolutionapparatus: Paddle at 100 rpm).

FIG. 17 shows the dissolution profile APAP (˜335 mg) capsules (size #0),sealed with HPMC-6 mg/cm2) and coated with EUDRAGIT®—L100/S100 (75/25) 5mg/cm² and 7.5 mg/cm² in multi-media. (USP dissolution apparatus: paddleat 100 rpm).

FIG. 18 shows the dissolution profile of APAP Capsule-in-capsule (CIC)[Inner capsule (size #3) band sealed and coated with 10 mg/cm²EUDRAGIT®-EPO & Outer capsule (size #0), band sealed and coated with 5mg/cm² EUDRAGIT®-L100/S100 (75/25)] in multimedia. (USP dissolutionapparatus—paddle at 100 rpm; n=6).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ various conventionaltechniques of molecular biology (including recombinant techniques),microbiology, cell biology, biochemistry, nucleic acid chemistry, andimmunology, which are within the skill of the art. Unless indicatedotherwise, the following terms have the following meanings when usedherein and in the appended claims. Those terms that are not definedbelow or elsewhere in the specification shall have their art-recognizedmeaning.

A “stable” formulation or composition is one in which the biologicallyactive material therein essentially retains its physical stability,chemical stability, and/or biological activity upon storage. Stabilitycan be measured at a selected temperature and humidity conditions for aselected time period. Trend analysis can be used to estimate an expectedshelf life before a material has actually been in storage for that timeperiod. For live bacteria, for example, stability may be defined as thetime it takes to lose 1 log of CFU/g dry formulation under predefinedconditions of temperature, humidity and time period.

“Viability” with regard to bacteria, refers to the ability to form acolony (CFU or Colony Forming Unit) on a nutrient media appropriate forthe growth of the bacteria. Viability, with regard to viruses, refers tothe ability to infect and reproduce in a suitable host cell, resultingin the formation of a plaque on a lawn of host cells.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” may in certain instances refer to lowering of an event orcharacteristic (e.g., microorganism growth or survival). It isunderstood that this is typically in relation to some standard orexpected value, in other words it is relative, but that it is not alwaysnecessary for the standard or relative value to be referred to. Forexample, “reduces the population of bacteria” in certain instances mayrefer to lowering the amount of bacteria relative to a standard or acontrol.

By “treat” or other forms of the word, such as “treated” or “treatment,”may, in certain instances mean to administer a composition or to performa method in order to reduce, prevent, inhibit, break-down, or eliminatea particular characteristic or event (e.g., microorganism growth orsurvival).

The term “viable cell” may in certain instances mean a microorganismthat is alive and capable of regeneration and/or propagation, while in avegetative, frozen, preserved, or reconstituted state.

The term “viable cell yield” or “viable cell concentration” may, incertain instances refer to the number of viable cells in a liquidculture, concentrated, or preserved state per a unit of measure, such asliter, milliliter, kilogram, gram or milligram.

The term “cell preservation” in certain instances may refer to a processthat takes a vegetative cell and preserves it in a metabolically inertstate that retains viability over time. As used herein, the term“product” in certain instances may refer to a microbial composition thatcan be blended with other components and contains specifiedconcentration of viable cells that can be sold and used.

The terms “microorganism” or “microbe” in certain instances may refer toan organism of microscopic size, to a single-celled organism, and/or toany virus particle. The definition of microorganism used herein includesBacteria, Archaea, single-celled Eukaryotes (protozoa, fungi, andciliates), and viral agents.

The term “microbial” in certain instances may refer to processes orcompositions of microorganisms, thus a “microbial-based product” is acomposition that includes microorganisms, cellular components of themicroorganisms, and/or metabolites produced by the microorganisms.Microorganisms can exist in various states and occur in vegetative,dormant, or spore states. Microorganisms can also occur as either motileor non-motile, and may be found as planktonic cells (unattached),substrate affixed cells, cells within colonies, or cells within abiofilm.

The term “prebiotic” in certain instances may refer to food ingredientsor bacterial producing ingredients that are not readily digestible byendogenous host enzymes and confer beneficial effects on an organismthat consumes them by selectively stimulating the growth and/or activityof a limited range of beneficial microorganisms that are associated withthe intestinal tract. Also the term includes one or more livemicroorganisms that confer beneficial effects on a host organism.Benefits derived from the establishment of probiotic microorganismswithin the digestive tract include reduction of pathogen load, improvedmicrobial fermentation patterns, improved nutrient absorption, improvedimmune function, improved intestinal hormonal signaling and metabolicregulation, aided digestion and relief of symptoms of irritable boweldisease and colitis.

The term “Symbiotic” in certain instances may refer to a compositionthat contains both probiotics and prebiotics. Symbiotic compositions arethose in which the prebiotic compound selectively favors the probioticmicroorganism.

The term “gastrointestinal tract” in certain instances may refer to thecomplete system of organs and regions that are involved with ingestion,digestion, and excretion of food and liquids. This system generallyconsists of, but not limited to, the mouth, esophagus, stomach and orrumen, intestines (both small and large), cecum (plural ceca),fermentation sacs, and the anus.

The term “pathogen” in certain instances may refer to any microorganismthat produces a harmful effect and/or disease state in a human or animalhost.

The pharmaceutical formulations provided herein may further include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable buffered saline solutionssuch as phosphate buffered saline solutions pH 7.0-8.0. Suitablepharmaceutical carriers include, but are not limited to sterile water,salt solutions (such as Ringer's solution), alcohols, polyethyleneglycols, gelatin, carbohydrates such as lactose, amylose or starch,magnesium stearate, talc, silicic acid, viscous paraffin, fatty acidesters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. Thepharmaceutical preparations can be mixed with auxiliary agents, e.g.,lubricants, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, coloring, and/or aromaticsubstances and the like which do not deleteriously react with the activecompounds. They can also be combined where desired with other activesubstances, e.g., ileal brake hormone regulatory substances to improvemetabolism and ameliorate metabolic syndromes.

Compounds provided herein may be formulated in a pharmaceuticalcomposition, which may include pharmaceutically acceptable carriers,thickeners, diluents, buffers, surface active agents, neutral orcationic lipids, lipid complexes, liposomes, penetration enhancers,carrier compounds and other pharmaceutically acceptable carriers orexcipients and the like in addition to the compound.

Pharmaceutical compositions may also include one or more activeingredients such as, anti-inflammatory agents, anesthetics, and thelike. Formulations for oral or intravaginal administration may includebuffers, liposomes, diluents and other suitable additives. Thecompositions provided herein may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional compatible pharmaceutically-active materials such as,e.g., statins, linaclotide, ileal brake hormone releasing substances,anti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, antioxidants,opacifiers, thickening agents and stabilizers. Depending on theparticular active ingredients, the formulations may be administered inthe same pill or tablet or as a distinct pill or tablet as part of aco-administration protocol. However, such materials, when added, shouldnot unduly interfere with the biological activities of the components ofthe compositions provided herein.

Regardless of the method by which compounds are introduced into apatient, colloidal dispersion systems may be used as delivery vehiclesto enhance the in vivo stability of the compounds and/or to target thecompounds to a particular organ, tissue or cell type. Colloidaldispersion systems include, but are not limited to, macromoleculecomplexes, nanocapsules, microspheres, beads and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, liposomesand lipid:compound complexes of uncharacterized structure. A preferredcolloidal dispersion system is a plurality of liposomes. Liposomes aremicroscopic spheres having an aqueous core surrounded by one or moreouter layers made up of lipids arranged in a bilayer configuration (see,generally, Chonn et al., Current Op. Biotech. 6, 698-708 (1995)).Likewise, microparticulate or nanoparticulate polymeric bead dosageforms may be used in composition provided herein. Compounds providedherein may be used in combination with one or more additional activeagent and encapsulated in a particulate dosage form. In this manner,certain compounds provided here, alone or in combination with otheractive agents, are released at that site over time to provide asustained therapeutic benefit. Release of the active agent from theparticulate dosage forms of the present invention can occur as a resultof both diffusion and particulate matrix erosion. Biodegradation ratedirectly impacts active agent release kinetics.

In preferred embodiments, the pharmaceutical composition of theinvention is administered orally. Dosing can be dependent on a number offactors, including severity and responsiveness of the disease state tobe treated, and with the course of treatment lasting from several daysto several months, or until a cure is effected or a diminution of thedisease state is achieved. Toxicity and therapeutic efficacy ofcompounds provided herein can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals. For example, fordetermining The LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds whichexhibit large therapeutic indices are preferred. While compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissues in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

The data obtained from in vitro and in vivo assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of exposureconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a local exposure ranges thatincludes the cell derived IC₅₀ (i.e., the concentration of the testcompound which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma areexpected to be unmeasurably low. Dosing schedules can be calculated frommeasurements of drug accumulation in the intestinal tract and feces ofthe patient. Not relevant, organisms are not absorbed.

Suitable dosage amounts for probiotic organisms may, for example, varyfrom about 10⁵ to 10¹² organisms, typically about 10⁶ based on thenumbers of organisms found in the ileum of said patient. Similarly,delivery of compounds provided herein will be specific to particularcells, conditions, and locations, such as ileum. In general, dosage isfrom tablets, capsules, granules and microgranules, powders, liquids andalike, and which may be given once or more daily, weekly, monthly oryearly, or even less frequently. In the treatment or prevention ofcertain conditions, an appropriate dosage level will generally be asabove per day which can be administered in single or multiple doses.Live microorganisms or therapeutic compounds according to the invention(e.g. live organisms) may be formulated into pharmaceutical compositionsfor administration according to known methodologies, including forexample using immediate-release, as well as pulsatile-release, anddelayed-release technologies. Pharmaceutical compositions may, forexample, comprise one or more constructs, in combination with apharmaceutically acceptable carrier, excipient or diluent. Such carrierswill be nontoxic to recipients at the dosages employed. A suitabledosage may be from about as above, per species at least 10⁵ to 10¹² oraland various ranges within these amounts being still more typical foradministration. It will be evident to those skilled in the art that thenumber and frequency of administration will be dependent upon theresponse of the host. “Pharmaceutically acceptable carriers” fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, saline andphosphate-buffered saline at physiological pH may be used. Stabilizers,dyes and even flavoring agents may be provided in the pharmaceuticalcomposition.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compounds of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

However, pharmaceutical compositions provided herein may be in any formwhich allows for the composition to be administered to a patient by theoral route and less commonly by intravaginal or rectal routes. Thepharmaceutical composition is formulated so as to allow the activeingredients contained therein to be bioavailable at the site targetedupon administration of the composition to a patient. Compositions thatwill be administered to a patient take the form of one or more dosageunits, where for example, a tablet may be a single dosage unit, and acontainer of one or more compounds of the invention in oral form mayhold a plurality of dosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed,applying common membranes used for microencapsulation and suitable forthe microencapsulation of live probiotic organisms include biodegradablesynthetic “polymers” such as polylactide, polyglycolic acid, andpolyanhydride. Established “polymers” for live encapsulation and enzymeencapsulation include alginate-polylysine-alginate (APA),alginate-polymethylene-co-guanidine-alginate (A-PMCG-A),hydroymethylacrylate-methyl methacrylate (HEMA-MMA), MultilayeredHEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC),acrylonitrile/sodium methallylsulfonate (AN-69), polyethyleneglycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane(PEG/PD₅/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), Siliceousencapsulates and cellulose sulphate/sodiumalginate/polymethylene-co-guanidine (CS/A/PMCG). Other materials thatare useful include, without limitation, cellulose acetate phthalate,calcium alginate and k-carrageenan-Locust bean gum gel beads,gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starchpoly-anhydrides, starch polymethacrylates, polyamino acids, entericcoating polymers.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: diluents such as water, preferably fixed oilssuch as synthetic mono or diglycerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose.

Compounds described herein can be used in diagnostics, therapeutics,prophylaxis, and as research reagents and in kits. Provision of meansfor detecting compounds of the invention can routinely be accomplished.Such provision may include enzyme conjugation, radiolabelling or anyother suitable detection systems. Kits for detecting the presence orabsence of compounds of the invention may also be prepared.

The compounds of the invention may also be used for research purposes.Thus, the specific activities or modalities exhibited by the compoundsmay be used for assays, purifications, cellular product preparations andin other methodologies which may be appreciated by persons of ordinaryskill in the art.

Using the Smart Pill to study pH of the intestinal tract and therebydefine the pH of the target sites of ileum and colon for release ofspecific probiotic organisms. Recently, the SmartPill, a wirelesspH/pressure recording capsule, has been utilized to measure the wholegut transit time. Wireless capsule motility, using the SmartPill GImonitoring system, samples and transmits intraluminal pH, pressure, andtemperature data from a capsule at regular intervals as it traversesthrough the gastrointestinal tract; from these, gastric emptying andwhole gastrointestinal tract transit can be assessed. In addition, thereare a few studies on the small bowel pH. The aim of this study was toinvestigate the relationship between small bowel disease and the smallbowel pH, using the SmartPill to non-invasively record sequential imagesand the pH. Volunteers swallowed the SmartPill with 240 mL of water. TheSmartPill transmitted the acquired images and the pH to the recorderunit located outside the body for about ten hours while the subject wasfasting. SmartPill capsule shows promise as a useful diagnostic test toevaluate patients for GI transit disorders and to study the effects ofdiseases of the gastrointestinal tract on pH and GI transit (8). Theintragastric pH was low and after gastric emptying the pH in the wholesmall intestine rose from 6.0 to as high as 8.1 in the ileum, then afterpassing the ileocecal valve, the pH of the right colon was once again5.5 to 6.5. The pH value increased from the duodenum to the terminalileum (p<0.0001) in all patients, but diabetic subjects and obesepatients did not rise as high as normal subjects. These findings wereunexpected, and indicate that the release target is different forformulated probiotic organisms in the ileum and right colon of diabeticsand obese patients, compared to normal subjects. Clearly, effectivepractice of site specific delivery in the human intestinal tractrequires adjustment for the differing conditions of the local ileummicro-environment, surprisingly a feature of disease associated changesin the microbiome as taught by experiments using SmartPill. Thus theconcept of targeted probiotic replacements and making changes insignaling processes to treat disease is advanced to practice. Probioticformulations and dosages and compositions must be completely changed todeal with these new discoveries.

Methods

Metabolic Syndrome and Obesity

There were two organisms that were found in intestinal flora in minornumbers, but which represented major regulatory balance organisms in thedevelopment of obesity and associated metabolic abnormalities,Methanobrevibacter smithii which promotes adiposity and Bacteroidestheataiotaomicron, which down regulates metabolic syndrome associatedinflammation and thereby removes the associated risk to thecardiovascular integrity of the host (1). Another organism found byothers to be important is Faecalobacterium prausnitzii, the absence ofwhich appears to correlate with worsening of obesity and Type 2 diabetes(2). In the practice of the instant invention, this organism is a targetfor replacement via targeted delivery to the ileum as in the presentinvention.

Resident host microflora condition and prime the immune system in thepreferred practice of the invention are disclosed herein. However,systemic and mucosal immune responses to bacteria may be divergent.Several workers have examined the relationships between the immunesystem and the microbiome components in the gastrointestinal tract. Forexample, our work with patients having RYGB surgery showed elevatedendotoxin and high levels of inflammation prior to surgery, followedafter surgery by remediation and a lowering of inflammatory processes.(3). It is important to understand that current viewpoints show theileum and the ileal brake hormone pathways to be the beneficial site ofaction of RYGB surgery, which is an effective treatment for obesity andin fact the only known means of curing metabolic syndrome associatedtype 2 diabetes. It is shown herein that the actions are mediated at thelevel of the ileum and the ileal brake, and the novel discovery was alowering of chronic inflammation, presumed a cause of revised microbiomeand revised signaling at the level of the intestinal L-cells. Additionalnovel discovery was the level of close interaction between theintestinal L-cells, the intestinal bacteria, and the systemic hostinflammation, which is responsible for the various diseases that areconsidered part of overall metabolic syndrome in humans (3). O'Mahonyand colleagues examined the inflammatory signaling processes involved inthis pathway. Their aim was to compare, in vitro, cytokine production byhuman mononuclear and dendritic cells (DCs) from mesenteric lymph nodes(MLNs) and peripheral blood mononuclear cells (PBMCs) to definedmicrobial stimuli. Mononuclear cells and DCs isolated from the MLN(n=10) and peripheral blood (n=12) of patients with active colitis wereincubated in vitro with the probiotic bacteria Lactobacillus salivariusUCC118 or Bifidobacterium infantis 35624 or the pathogenic organismSalmonella typhimurium UK1. Interleukin (IL)-12, tumor necrosis factor(TNF)-alpha, transforming growth factor (TGF)-beta, and IL-10 cytokinelevels were quantified by ELISA. PBMCs and PBMC-derived DCs secretedTNF-alpha in response to the Lactobacillus, Bifidobacteria, andSalmonella strains, whereas MLN cells and MLN-derived DCs secretedTNF-alpha only in response to Salmonella challenge. Cells from thesystemic compartment secreted IL-12 after co-incubation with Salmonellaor Lactobacilli, whereas MLN-derived cells produced IL-12 only inresponse to Salmonella. PBMCs secreted IL-10 in response to theBifidobacterium strain but not in response to the Lactobacillus orSalmonella strain. However, MLN cells secreted IL-10 in response toBifidobacteria and Lactobacilli but not in response to Salmonella. Inconclusion, commensal bacteria induced regulatory cytokine production byMLN cells, whereas pathogenic bacteria induce T cell helper 1-polarizingcytokines. Commensal-pathogen divergence in cytokine responses is moremarked in cells isolated from the mucosal immune system compared withPBMCs (4). This work indicates that the endogenous cellular signalingpathways at work in the distal gastrointestinal tract can discriminatetheir responses as the flora in the microbiome change between commensalsand pathogens.

Immunoregulatory Pathways

Leukocyte recruitment is a central immune process. Multiple factors havebeen described to promote leukocyte infiltration into inflamed tissues,but only recently has evidence for endogenous negative modulators ofthis inflammatory process emerged. The discovery of several locallyproduced modulators has emerged into a new field of endogenousinhibitors of leukocyte extravasation. Recent findings from severalinflammatory disease models show that tissues can self-regulate therecruitment of inflammatory cells, suggesting that local tissues mayhave a greater ‘regulatory say’ over the immune response than previouslyappreciated (5). Organisms targeted for replacement in obese or diabeticpatients could be delivered as components of an oral site specificdelivery formulation designed to assist in the management of metabolicsyndrome and prevent or control associated inflammatory manifestationssuch as obesity and type 2 diabetes. This novel therapeutic approach,based on changing local signaling at the level of intestinal L-cells anddendritic cells, is proposed based on the observation that locallyproduced modulators of leukocyte recruitment may represent localhomeostatic mechanisms that tissues and organs may have evolved forprotection against the destructive potential of the immune system (5).The involvement of the local microbiome flora as a protective factor,beneficial to the host, is a novel aspect in the practice of theinvention, since this would explain why use of certain antibiotics usedfor treatment of infection may cause more problems from dysbiosis thanare solved by eradicating pathogens.

Larsen and colleagues have studied the link between metabolic diseasesand bacterial populations in the gut. The aim of their studies was toassess the differences between the compositions of the intestinalmicrobiota in humans with type 2 diabetes and compare to controls whowere non-diabetic persons. The study population included 36 male adultswith a broad range of age and body-mass indices (BMIs), among which 18subjects were diagnosed with diabetes type 2. The fecal bacterialcomposition was investigated by real-time quantitative PCR (qPCR) and ina subgroup of subjects (N=20) by tag-encoded amplicon pyrosequencing ofthe V4 region of the 16S rRNA gene. The proportions of phylum Firmicutesand class Clostridia were significantly reduced in the diabetic groupcompared to the control group (P=0.03). Furthermore, the ratios ofBacteroidetes to Firmicutes as well as the ratios ofBacteroides-Prevotella group to C. coccoides-E. rectale group correlatedpositively and significantly with plasma glucose concentration (P=0.04)but not with BMIs. Similarly, class Betaproteobacteria was highlyenriched in diabetic compared to non-diabetic persons (P=0.02) andpositively correlated with plasma glucose (P=0.04). The results of thisstudy indicated that type 2 diabetes in humans is associated withcompositional changes in intestinal microbiota. The level of glucosetolerance should be considered when linking microbiota with metabolicdiseases such as obesity and developing strategies to control metabolicdiseases by modifying the gut microbiota (6).

Recent studies have focused additional attention on intestinalmicrobiota as environmental factors that increase energy yield fromdiet, regulate peripheral metabolism and thereby increase body weight.Obesity is associated with substantial changes in composition andmetabolic function of gut microbiota, but the pathophysiologicalprocesses driving this bidirectional relationship have not been fullyelucidated. Clearly there are important relationships between thecomposition of gut microbiota, energy extracted from diet, synthesis ofgut hormones involved in energy homeostasis, production of butyrate andthe regulation of fat storage (7). The most important discoveries ofthis work are from our own studies examining the release of hormonesfrom the distal intestines in response to stimulating factors such asfoods and probiotic organisms (3).

Regulation of the Host Metabolome and the Invention of the MetabolomicMetaSensor

The distal intestine's responsiveness to molecules presented to it viadiet is important in regulating the upstream sensory drivers ofingestion such as hunger, taste, smell, and appetite. Together theinteraction between ingestion, selective absorption and feedbackregulatory control of appetite ensure that the organism is properlynourished and its energy needs are properly balanced by intake of foodas fuel, and the current term used to describe the steps in theseprocesses is Metabolomics. It is important to consider the “organism” inthis case to be the combination of all cells, including bacteria,viruses, fungi and human cells, together a MetaOrganism, which in termsof cell numbers is more than 90% non-human cells. The biosensorseffecting these complex processes are interactive between the non-humancells and the human cells, together a MetaSensor, and it is expectedthat most Metabolomic processes will be controlled by MetaSensorysignaling, i.e. interactiveness between non-human and human cells.Likewise, it is theorized that host immunity and thus conditions likefood allergy are controlled by these same distal intestinal MetaSensors.Considering the central role of the master regulatory MetaSensor in hostmetabolomics at homeostasis, it is clear that food intake is regulatedby the combined sensor signals defining input (brain programmed appetiteinteracting with taste and smell), counterbalanced precisely by thedistal intestinal sensor signals such as the ileal brake and associatedhormonal regulatory “stop input” signals that are received wheningestion exceeds the ability of the organism to absorb upstream in theduodenum and jejunum. In normal operations, host demand for energydominates, and ingestion of nutrients proceeds unimpeded by stopsignals. When energy intake exceeds demand, there is both short term andlong term storage of excess. Short term storage includes abdominaladipose and liver, and long term storage is peripheral adipose, both ofwhich interact with the MetaSensory signaling processes to balancesupply and demand for energy. There is good understanding of the balancecreated between ingestion and storage, driven largely by appetite andthe combined sensory input from taste and smell. However, it is novel toidentify the ileal brake and its associated regulatory MetaSensorcomponent as a stop signal on the ingestion process, and our work inthis area with the pathways operative in RYGB patients is illustrative(see WO2012-118712, hereby incorporated in its entirety), wherein wehave shown that ileal delivery of food substances creates a stop signalbecause the MetaSensor detects malabsorption and uses the hormonesreleased from the L-cells of the ileum (GLP-1, PYY and many others) toshut down ingestion and appetite via brain stimulatory feedback. In abreakthrough discovery, we are now advancing a plausible means ofoperating the ileal brake component of the intestinal MetaSensor, andits integral controllers. In an individual of normal weight and innutritional balance the ileal brake component of the MetaSensor, (thecontroller of the stop signal) is comprised of host L-cells interactingwith beneficial intestinal organisms. The intestinal bacteria are anessential component of MetaOrganism Metabolomics, and it is logical forthem to have a major regulatory role in the operation of the stop signalfrom the ileum. Intestinal microbes lack the ability to signal the hostbrain directly, so they use host signaling pathways to make their needsknown. The combined MetaSensor operates in the distal intestine, viainteraction with the L-cells to regulate the stop signal to mutualbenefit. Described simply, certain intestinal bacteria can suppress thestop on the appetite signal from the brain. They do this when they arehungry for a nutrient, food or even a specific molecule. When microbesdeep in the intestinal tract are hungry, the host is hungry because thestop signal of the MetaSensor is inactivated. FIGS. 1 to 7 diagram theMetaSensor in detail, and show how it is comprised overall in FIG. 1,and in FIGS. 2-7 describe its operations in the ileum that controlmetabolomics and immunity. The MetaSensor in the ileum producesregulatory hormonal output from the combined actions of the enteralL-cells, and the intestinal bacteria. FIG. 2 shows normal operations ofthe MetaSensor via stop signals GLP-1, PYY and other L-cell derivedregulatory hormones. Notably the system is in balance when diet isbalanced and thus some excess reaches the distal intestine. However,when the patient ingests only IR-CHOs, the bacteria in the ileum are notachieving nutrition. They react by Suppression of L-cell output andhunger ensues. If on the other hand the patient is having a balance dietwith portions reaching the bacteria, they have no reason to suppress theL-cell output and normal eating produces satiety. FIG. 3 shows thesituation when a diabetogenic food, such as sugar sweetened beveragealters the microbiome and thus the hormonal operation of the MetaSensor.FIG. 4 shows the situation when there is a Microbiome dysbiosis thatproduces abnormal regulatory control of the MetaSensor via its action onthe L-cells. With reference to our previous work with the ileal brakeoperations in health and disease, FIG. 5 shows the impact of RYGBsurgery on the MetaSensor. Notably, RYGB surgery mechanically divertsingested contents past the absorptive (but non-signaling) area, andbombards the signaling areas further downstream in late jejunum andileum. The arrival of massive nutrients at the ileum in such a largequantity creates a “malabsorptive emergency” and initiates the satietysignal by shutting down the hormonal release from the L-cells toregenerate signaling to a certain extent with the same or less amount offood needed, therefore restoring maintenance and regeneration. Andbecause it is not individualized, RYGB surgery will trigger moreregeneration than signaling to the point that about 4 years down theline, the jejunum will evolve to restore absorption to a baselinelevels. FIG. 6 shows the impact of an oral mimetic of RYGB, an ilealbrake hormone releasing substance called Brake, on the MetaSensor. Brakeacts the distally in the same way as RYGB surgery. There is the sameactivation of L-cells, the output of which produce regeneration and makehunger disappear into satiety. The strength of the ileal signal is notas potent as RYGB, but it can be more prolonged because of the delayedrelease formulation. Thus with Brake, the intensity of the stimulationwill be more moderate and closer to physiological and thereforeregeneration proceeds in Liver, pancreas, GI enterocytes in a much morenatural and physiological way compared to surgery. Of no great surprise,weight loss is more rapid with RYGB, since RYGB surgery also physicallydecreases the size of stomach, limiting ingestion in a second, profoundmanner over the ileal brake pathway alone. Finally, FIG. 7 considers theimpact of a common diabetes drug, metformin, in combined with Brake, onthe operations of the MetaSensory process, illustrating synergisticinteractions between a drug and a mimetic of RYGB surgery, this exampleis illustrative, and there are many more of these with other drugs usedin the treatment of metabolic syndrome manifestations such as type 2diabetes.

Briefly, the MetaSensor gives the stop signal to the brain via ilealbrake hormones in response to its detection of perceived malabsorption.In total, the novel aspect of the invention, shown by this discussionand these figures, is the nature of this MetaSensors action on the hostmetabolome, that effect being the combined action of L-cell output fromdetection of food delivery and the actions of probiotic organisms on theL-cells to modify the signal in response to nutritional demandscommunicated by bacteria. It is remarkable how effectively the probioticorganisms control our appetite and selection of nutrients and foods tosuit their own purposes. We are together with our probiotic symbionts, abalanced ecosystem, a true MetaOrganism. In homeostasis, all parties aresuccessfully meeting their needs. Diseases, all loosely described ascomponents of Metabolic Syndrome, are the results of imbalances, whichmay be bacterial in origin, or arise from the cells of the host.Regardless, both components of the MetaSensor must receive therapeuticattention if homeostasis is to be restored, and the current submissionprovides detailed means of re-balancing the MetaSensory output torestore homeostasis and remove diseases. All of the therapeutic advancesdescribed herein, and those to follow ongoing research, aretransformational steps mediated by treatments changing the input-outputproperties of the MetaSensor described herein.

There are some other useful aspects to the MetaSensor in the distalileum. Specifically, the MetaSensor provides a quick immune systemmediated response when a foreign invader is detected in the GI tractlumen, and the rapid improvement of intestinal dysbiosis such asinfection with C. difficile can be remediated by replacement of C.difficile with more beneficial organisms delivered by formulation to theileum and colon via the oral formulations described herein. Furthermore,there are preferred enablements as distal ileum vaccines that are orallyactive. Specific examples are found in PCT/US13/31483, the entirety ofwhich is herein incorporated by reference.

In parallel with the stimulation of the MetaSensor with a foreignorganism, it is now clearly apparent that the MetaSensor is responsiveto chemical substances that act on the probiotic bacteria, each of whichhas a specific molecule that excites a response which is thencommunicated to the human host via the L-cells, lymphoid tissue inPeyer's Patches, or in all possibility any enterocyte found in thelumen. When the host or the integrated intestinal organisms of theMetaOrganism encounters a deficiency of any nutritional component oressential substance, the signal for this deficiency comes to the brainfrom the MetaSensor in the intestine (if communicated by the hostmicrobiome), and perhaps from the brain or tongue or nose ifcommunicated by the host cells. The actions of the host to obtain thatmissing substance are perceived as “cravings” and after satisfied theMetaSensor stops the search. Thus, when the Microbiome organisms arehungry for something specific, we as the MetaOrganism are instructed toobtain that specific substance. This novel discovery immediately opensopportunities to regulate ingestion of potentially harmful substanceslike refined sugar, via therapeutic strategies focused on the MetaSensoritself, and explains the ability of ileum delivered glucose to regulatediabetes and other diseases called metabolic syndrome (see WO2010-027498 and WO 2013-063527 A1, herein incorporated by reference).While these inventions focus on the needs of the host via regulatoryMetaSensor action, it can readily be seen that regulating the Microbiomevia targeted replacement will also impact the diseases of the host in abeneficial way. Replacement of organisms missing in association withmetabolic diseases such as obesity, for example faecalibacteriumprausnitzii, is now possible with the ability disclosed herein toprovide targeted oral delivery of live organisms to the site of theMetaSensor in the ileum.

The following Examples are offered by way of illustration and not by wayof limitation.

Example 1

Example 1 is directed toward the making and testing of a formulationaccording to the invention for the treatment of a Clostridium difficileinfection.

Biological Assays

Standard therapies for antibiotic-associated diarrhea (AAD) andClostridium difficile-associated diarrhea (CDAD) have limited efficacy.Probiotic prophylaxis is a promising alternative for reduction of AADand CDAD incidence. The preferred embodiment is microgranulesadministered to said patient with Clostridium difficile infection isabout 10⁵ to 10¹² cfu of one or more species of probiotic organisms,targeted to ileum and ascending colon. Preferred embodiments would be amixture of probiotic organisms reflective of the balance and componentsof the microbiome of a normal human subject, preferably a patient freeof antibiotic exposure in the past and not infected with C. difficileorganisms. The clinical protocol for testing the efficacy of saidformulation would administer said formulation of probiotic organisms topatients in the manner followed by others who have tested theeffectiveness of probiotics or fecal transplantation for infections withClostridium difficile in human patients. By way of example, asingle-center, randomized, double-blind, placebo-controlled dose-rangingstudy, is conducted for one of these probiotics in adult inpatientsallocated to one of three groups: two probiotic capsules per day, oneprobiotic capsule and one placebo capsule per day, or two placebocapsules per day. In the design using un-protected formulations of eachprobiotic organism, each probiotic capsule contained 50 billion c.f.u.of live organisms. Probiotic prophylaxis or treatment began within 36 hof initial antibiotic administration, continued for 5 days after thelast antibiotic dose, and patients were followed for an additional 21days. In this study, Pro-2 (15.5%) had a lower antibiotic associateddiarrhea (AAD) incidence vs. Pro-1 (28.2%). Each probiotic group had alower AAD incidence vs. placebo (44.1%). In patients who acquired AAD,Pro-2 (2.8 days) and Pro-1 (4.1 days) had shorter symptom duration vs.placebo (6.4 days). Similarly, Pro-2 (1.2%) had a lower Clostridiumdifficile associated diarrhea (CDAD) incidence vs. Pro-1 (9.4%). Eachtreatment group had a lower CDAD incidence vs. placebo (23.8%).Gastrointestinal symptoms were less common in the treatment groups vs.placebo and in Pro-2 vs. Pro-1. The proprietary probiotic blend used inthis study was well tolerated and effective for reducing risk of AADand, in particular, Clostridium difficile associated diarrhealinfections in hospitalized patients on antibiotics. A dose-rangingeffect was shown with 100 billion c.f.u., yielding superior outcomes andfewer gastrointestinal events compared to 50 billion c.f.u. (9).Clearly, a protective formulation would allow the targeted delivery ofsmaller numbers of these organisms, lowering the costs of production ofthe organisms for the product.

In follow up to the study design above, Johnson and colleagues conducteda literature search for randomized, placebo-controlled efficacy studiesof probiotic use among adults receiving antibiotics, in whichClostridium difficile infection (CDI) was one of the outcomes measured.In addition, they conducted meta-analyses of probiotics that wereincluded in more than one randomized trial. Eleven studies wereidentified; most were seriously underpowered to determine the efficacyof probiotics in the prevention of CDI. Two showed significantly lowerrates of CDI among the probiotic recipients. A meta-analysis of threestudies that used the probiotic combination Lactobacillus acidophilusCL1285 and Lactobacillus casei LBC80R and a combined analysis of thosestudies with four studies that used Saccharomyces boulardii, showedlower CDI rates in recipients of probiotics compared with recipients ofplacebo (risk ratio=0.39; 95% confidence interval 0.19-0.79). Thus,while potential flaws in study design were identified, a review of theavailable literature suggested that the primary prevention of CDI withspecific probiotic agents may be achievable. Additional studies ofsufficient size and with rigorous design are needed to confirm thesefindings. (10) By way of commentary, the studies reviewed did not targetthe probiotic organisms, and thus the present invention is far moreeffective than those unprotected formulations used thus far.

Materials and Methods:

Described below are formulations that are being made and tested for thetarget delivery for testing in biological assays, the formulation havingan antibiotic (Vancomycin 250 mg) (millimeter range) for release at pH1.0-6.0 in stomach, duodenum and ileum and symbiotic (prebiotic:L-Leucine; probiotic: species of: lactobacillus and bifidobacterium) forrelease at pH 5.5-6.2 in right colon every 6 hours.

Active Pharmaceutical Ingredient (API):

-   -   Antibiotic—Vancomcyin hydrochloride (micronized) supplied by        local generic US/non-US suppliers, e.g., LGM Pharma, USA, etc.    -   Prebiotics—proteins (casein, hydrolyzed protein, etc.),        peptides, amino acids (L-Leucine), carbohydrates glucose,        lactose, starches, inulin, etc. and certain bacterial strains:        provided by Denisco, CHR Hansen, Institu Risell—Lallemand and        other high quality global suppliers of prebiotics.    -   Live probiotics Species of: lactobacillus and bifidobacterium        provided by Denisco, CHR Hansen, Institu Risell—Lallemand and        other high quality global suppliers.    -   Inactive Ingredients (Excipients):    -   Microcrystalline, starch, HPMC or equivalent “polymers”, hard        gelatin capsules, and other fillers, etc.—purchased from local        US supplier such as FMC, Capsugel, Colorcon, etc.

Intermediate Formulation/Manufacturing Process (at local CMO):

“Uncoated Antibiotic Granules/Pellets” (millimeter range):

Ingredients Amount (%) Vancomcyin 50% Excipients (Microcrystallinecellulose - filler, 50% polyvinylpyrrolidone - binder, pregelatinizedstarch - disintegrant, silicon dioxide - flow aid, magnesium stearate -lubricant) Water as required 0%

Prepare a dry granulation with antibiotic and excipients in a low orhigh shear mixer and/or perform wet granulations with water and furtherpelletize using extruder/spheronizer and then drying to remove excesswater using optimized conditions “pH 5.0 to 6.0 Enteric Coated (EC)Antibiotic Granules/Pellets” (millimeter range):

Ingredients Amount (%) Uncoated Antibiotic Granules/Pellets 95% HPMC orequivalent “polymers” (Barrier and Seal coats) 1% “Polymers” (pH 5.0 to6.0 sensitive coating) 4% Water/Solvents as required 0%

The Uncoated Antibiotic Granules/Pellets are coated (the barrier coat)with aqueous or solvent coating solution of HPMC or equivalent“polymers” to coat in a coating pan or fluid bed drier/coater usingoptimized conditions. The barrier coated micropellets or granules arefurther coated with aqueous or solvent coating solution of pH 5.0 to6.0, sensitive coating “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The above pH 5.0 to 6.0,sensitive coated micropellets or granules are seal coated with aqueousor solvent coating solution of HPMC or equivalent “polymers” in acoating pan or fluid bed drier/coater using optimized conditions.

“pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pellets” (100micron).

Ingredients Amount (%) L. Leucine (prebiotic) 5% Freeze dried bacterial(species of lactobacillus and 1% bifidobacterium) probiotic) Excipients(Microcrystalline cellulose - filler, 82% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant, silicon dioxide - flow aid,magnesium stearate - lubricant) HPMC or equivalent “polymers” (Barrierand Seal coats) 2% “Polymers” (pH 5.5 to 6.2 sensitive coating) 10%Water/Solvents as required 0%

Prepared by dry granulating pre-/probiotic with excipients and/or wetgranulations with water solvents in high or low shear mixer and furtherpelletizing using extruder/spheronizer and then drying to remove waterusing optimized conditions. The above micropellets or granules arefurther coated (barrier coat) with aqueous or solvent coating solutionof HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The above barrier coatedmicropellets or granules are further coated with aqueous or solventcoating solution of “Polymers” (pH 5.5 to 6.2 sensitive coating) in acoating pan or fluid bed drier/coater using optimized conditions. Theabove pH 5.5 to 6.2 sensitive coated micropellets or granules are sealcoated with aqueous or solvent coating solution of HPMC or equivalent“polymers” in a coating pan or fluid bed drier/coater using optimizedconditions.

“pH 7.2 to 7.5 Enteric Coated (EC) Symbiotic Granules/Pellets” (100micron).

Ingredients Amount (%) pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic 88%Granules/Pellets HPMC or equivalent “polymers” (Seal coats) 2%“Polymers” (pH 7.2 to 7.5 sensitive coating) 10% Water/Solvents asrequired 0%

The above pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pelletsare coated with aqueous or solvent coating solution of “Polymers” (pH7.2 to 7.5 sensitive coating) in a coating pan or fluid bed drier/coaterusing optimized conditions. The above micropellets or granules arefurther coated with aqueous or solvent coating solution of HPMC orequivalent “polymers” (seal coat) in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Sachet—Formulation/Manufacturing Process (at Local CMO,Controlled Room and Humidity Conditions Throughout the Process):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 50% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 25% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 15% Excipients(Mannitol - filler, Silicon dioxide - glidant/flow aid) 10%

The above Uncoated Antibiotic Granules/Pellets, pH 5.0 to 6.0 EntericCoated (EC) Antibiotic Granules/Pellets and pH 7.2 to 7.5 Enteric Coated(EC) Symbiotic Granules/Pellets intermediate formulations are blended indesired portions in V-type or similar blender with excipients usingoptimized conditions. The blended powders are filling into sachets usingpowder filling equipment.

Example

Final Product—Capsules (Hard gelatin/HPMC)—Formulation/ManufacturingProcess (at local CMO, controlled room and humidity conditionsthroughout the process):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 20% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 12% Excipients(Microcrystalline cellulose - filler, Silicon dioxide - 13% glidant/flowaid) Hard Gelatin/HPMC Capsules 10%

The above Uncoated Antibiotic Granules/Pellets, pH 5.0 to 6.0 EntericCoated (EC) Antibiotic Granules/Pellets and pH 7.2 to 7.5 Enteric Coated(EC) Symbiotic Granules/Pellets intermediate formulations are blended indesired portions in V-type or similar blender with excipients. Theblended powders are filled into capsules using encapsulating equipment.

Example

Final Product—Capsules (Liquid Filled Hard or SoftGelatin)—Formulation/Manufacturing Process (at local CMO, controlledroom and humidity conditions throughout the process):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 35% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 10% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 10% Vegetable oil(immiscible liquid) 40% Gelatin as powder and Hard Gelatin Capsules 5%

The above Uncoated Antibiotic Granules/Pellets, pH 5.0 to 6.0 EntericCoated (EC) Antibiotic Granules/Pellets and pH 7.2 to 7.5 Enteric Coated(EC) Symbiotic Granules/Pellets intermediate formulations are blended indesired portions with immiscible liquid in a blender. Filled intocapsules using soft or hard gelatin encapsulating equipment usingoptimized conditions.

Example

Final Product—Capsule-in-Capsule (Hard gelatin) (1)Formulation/Manufacturing Process (at local CMO, controlled room andhumidity conditions throughout the process):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets 12% Uncoated Antibiotic Granules/Pellets 45% pH 5.0 to6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 25% Excipients(Microcrystalline cellulose - filler, Silicon dioxide - 10% glidant/flowaid) Small and Large Hard Gelatin/HPMC Capsules 8%

The pH 7.2 to 7.5 Enteric Coated (EC) Symbiotic Granules/Pelletsintermediate formulation is blended in with portion of excipients inV-type or similar blender and the blend. The blend is filled intosmaller capsules using encapsulating equipment and optimized conditions.The above Uncoated Antibiotic Granules/Pellets and pH 5.0 to 6.0 EntericCoated (EC) Antibiotic Granules/Pellets intermediate formulations areblended together in desired portions in V-type or similar blender withexcipients. The blended intermediate formulations along with the smallerfilled capsules are further filled into larger capsules usingspecialized capsule filling equipment and optimized conditions.

Example

Final Product—Capsule-in-Capsule (Hard gelatin)(2)—Formulation/Manufacturing Process (at local CMO, controlled room andhumidity conditions throughout the process):

Amount Ingredients (%) pH 5.5 to 6.2 Enteric Coated (EC) SymbioticGranules/Pellets 15% “Polymers” (pH 7.2 to 7.5 sensitive coating) 10%Uncoated Antibiotic Granules/Pellets 45% Excipients (Microcrystallinecellulose - filler, Silicon dioxide - 10% glidant/flow aid) pH 5.0 to6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 10% Small and LargeHard Gelatin/HPMC Capsules 10% Water/Solvents as required 0%

The pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pelletsintermediate formulation is blended in desired portions in V-type orsimilar blender with excipients. The blend is filled into smallercapsules using encapsulating equipment. The smaller filled capsules arefurther coated with pH 7.2 to 7.5 sensitive coating using aqueous orsolvent coating solution of “Polymers” in a coating pan or fluid beddrier/coater with optimized conditions. The above Uncoated AntibioticGranules/Pellets and pH 5.0 to 6.0 Enteric Coated (EC) AntibioticGranules/Pellets intermediate formulations are blended in desiredportions in V-type or similar blender with excipients. The smaller pH7.2 to 7.5 coated capsules and the blends are further filled into largercapsules using specialized capsule filling equipment and optimizedconditions.

Example

Final Product—Tablets/Microtablets—Formulation/Manufacturing Process (atlocal CMO, controlled room and humidity conditions throughout theprocess):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 12% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 12% Excipients(Microcrystalline cellulose - filler, 30% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant and silicon dioxide - flow aid,magnesium stearate - lubricant) HPMC or equivalent “Polymers” (Filmcoat) 1% Water/Solvents as required 0%

The Uncoated Antibiotic Granules/Pellets, pH 5.0 to 6.0 Enteric Coated(EC) Antibiotic Granules/Pellets and pH 7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets intermediate formulations are blended indesired portions in V-type or similar blender with excipients to aid inflow, disintegration and lubrication (for tableting machine). Theblended powders are compressed into Tablets/Microtablets using tabletingequipment. The tablets are further film coated using aqueous or solventcoating solution in a coating pan or fluid bed dryer using HPMC orequivalent “polymers” (Film coat).

Example

Final Product—Orally disintegrating Tablets(ODT)—Formulation/Manufacturing Process (at local CMO, controlled roomand humidity conditions throughout the process):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 12% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 12% Excipients(Mannitol - filler, polyvinylpyrrolidone - binder, 31% pregelatinizedstarch - disintegrant and silicon dioxide - flow aid, magnesiumstearate - lubricant)

The Uncoated Antibiotic Granules/Pellets, pH 5.0 to 6.0 Enteric Coated(EC) Antibiotic Granules/Pellets and pH 7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets intermediate formulations are blended indesired portions in V-type or similar blender with excipients. Theblended powders are compressed into soft tablets using tabletingequipment.

Example

Final Product—Tablet-in-Tablet (1)—Formulation/Manufacturing Process (atlocal CMO, controlled room and humidity conditions throughout theprocess):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 12% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 12% Excipients(Microcrystalline cellulose - filler, 30% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant and silicon dioxide - flow aid,magnesium stearate - lubricant) HPMC or equivalent “Polymers” (Filmcoat) 1% Water/Solvents as required 0%

The pH 7.2 to 7.5 Enteric Coated (EC) Symbiotic Granules/Pelletsintermediate formulation is blended in desired portions in V-type orsimilar blender with excipients to aid in flow, disintegration andlubrication (for tableting machine). The blended powders are compressedinto small tablets/Microtablets using tableting equipment. The aboveUncoated Antibiotic Granules/Pellets, and pH 5.0 to 6.0 Enteric Coated(EC) Antibiotic Granules/Pellets intermediate formulations are blendedin desired portions in V-type or similar blender with excipients to aidin flow, disintegration and lubrication (for tableting machine). Theblended powder is compress coated over the small tablets/Microtabletsusing compress coat tableting machine. The tablets are further filmcoated using aqueous or solvent coating solution in a coating pan orfluid bed dryer using HPMC or equivalent “polymers” (Film coat).

Final Product—Tablet-in-Tablet (2)—Formulation/Manufacturing Process (atlocal CMO, controlled room and humidity conditions throughout theprocess):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 12% pH 5.5 to 6.2Enteric Coated (EC) Symbiotic Granules/Pellets 12% Excipients(Microcrystalline cellulose - filler, 25% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant and silicon dioxide - flow aid,magnesium stearate - lubricant) “Polymers” (pH 7.2 to 7.5 sensitivecoating) 5% HPMC or equivalent “polymers” (Film coat) 1% Water/Solventsas needed 0%

The pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pelletsintermediate formulation is blended in desired portions in V-type orsimilar blender with excipients to aid in flow, disintegration andlubrication (for tableting machine). The blended powders are compressedinto small tablets/Microtablets using tableting equipment. Thecompressed tablets are coated with pH 7.2 to 7.5 sensitive coating usingaqueous or solvent coating solution of “Polymers” in a coating pan orfluid bed drier/coater with optimized conditions (“EC tablets”). Theabove Uncoated Antibiotic Granules/Pellets, and pH 5.0 to 6.0 EntericCoated (EC) Antibiotic Granules/Pellets intermediate formulations areblended in desired portions in V-type or similar blender with additionalexcipients to aid in flow, disintegration and lubrication (for tabletingmachine). The blended powder is compress coated over the small ECtablets/Microtablets using compress coat tableting machine. The tabletsare further film coated using aqueous or solvent coating solution in acoating pan or fluid bed dryer using HPMC or equivalent “polymers” (Filmcoat).

Example

Final Product—Tablet-in-Capsule (Hard gelatin)(1)—Formulation/Manufacturing Process (at local CMO, controlled room andhumidity conditions throughout the process):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 13% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 13% Excipients(Microcrystalline cellulose - filler, 24% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant and silicon dioxide - flow aid,magnesium stearate - lubricant) Hard Gelatin/HPMC Capsules 5%

The Uncoated Antibiotic Granules/Pellets, pH 5.0 to 6.0 Enteric Coated(EC) Antibiotic Granules/Pellets and pH 7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets intermediate formulations are blended indesired portions in V-type or similar blender with excipients to aid inflow, disintegration and lubrication (for tableting machine). Theblended powders are compressed into Tablets/Microtablets using tabletingequipment. The excipients and the tablets filled into hard gelatincapsules using specialized encapsulating equipment.

Example

Final Product—Tablet-in-Capsule (Hard gelatin)(2)—Formulation/Manufacturing Process (at local CMO, controlled room andhumidity conditions throughout the process):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 13% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 13% Excipients(Microcrystalline cellulose - filler, 24% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant and silicon dioxide - flow aid,magnesium stearate - lubricant) Hard Gelatin/HPMC Capsules 5%

The pH 7.2 to 7.5 Enteric Coated (EC) Symbiotic Granules/Pelletsintermediate formulations are blended in desired portions in V-type orsimilar blender with excipients to aid in flow, disintegration andlubrication (for tableting machine). The blended powders are compressedinto small tablets/Microtablets using tableting equipment. The aboveUncoated Antibiotic Granules/Pellets, and pH 5.0 to 6.0 Enteric Coated(EC) Antibiotic Granules/Pellets intermediate formulations are blendedin desired portions in V-type or similar blender with additionalexcipients to aid in flow, disintegration and lubrication (for tabletingmachine). The blended powder and tablets are filled into large HardGelatin Capsules using encapsulating equipment.

Example

Final Product—Tablet-in-Capsule (Hard gelatin)(3)—Formulation/Manufacturing Process (at local CMO, controlled room andhumidity conditions throughout the process):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 13% pH 5.5 to 6.2Enteric Coated (EC) Symbiotic Granules/Pellets 13% Excipients(Microcrystalline cellulose - filler, 20% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant and silicon dioxide - flow aid,magnesium stearate - lubricant) “Polymers” (pH 7.2 to 7.5 sensitivecoating) 4% Hard Gelatin/HPMC Capsules 5% Water/Solvents as required 0%

The pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pelletsintermediate formulations are blended in desired portions in V-type orsimilar blender with excipients to aid in flow, disintegration andlubrication (for tableting machine). The blended powders are compressedinto small tablets/Microtablets using tableting equipment. Thecompressed tablets are coated with pH 7.2 to 7.5 sensitive coating usingaqueous or solvent coating solution of “Polymers” in a coating pan orfluid bed drier/coater with optimized conditions (“EC tablets”). Theabove Uncoated Antibiotic Granules/Pellets, and pH 5.0 to 6.0 EntericCoated (EC) Antibiotic Granules/Pellets intermediate formulations areblended in desired portions in V-type or similar blender with excipientsto aid in flow, disintegration and lubrication (for tableting machine).The blended powder and the EC tablets are filled into a larger capsuleusing encapsulating equipment.

Example

Final Product—Bi-Layer Tablets—Formulation/Manufacturing Process (atlocal CMO, controlled room and humidity conditions throughout theprocess):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45% pH 5.0to 6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 13% Excipients(Microcrystalline cellulose - filler, 28% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant and silicon dioxide - flow aid,magnesium stearate - lubricant) pH 7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets 13% HPMC or equivalent “polymers” (Film coat)1% Water/Solvents as required 0%

The above Uncoated Antibiotic Granules/Pellets, and pH 5.0 to 6.0Enteric Coated (EC) Antibiotic Granules/Pellets intermediateformulations are blended in desired portions in V-type or similarblender with excipients to aid in flow, disintegration and lubrication(for tableting machine). The blended powders are compressed into tabletsusing bi-layer tableting equipment (“EC Tablets”). The pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets intermediate formulationsare blended in desired portions in V-type or similar blender withexcipients to aid in flow, disintegration and lubrication (for tabletingmachine). The blended powder is compressed over the EC tablets usingbilayer tableting machine. The tablets are further film coated usingaqueous or solvent coating solution in a coating pan or fluid bed dryerusing HPMC or equivalent “polymers” (Film coat).

Example

Final Product—Tri-Layer Tablets—Formulation/Manufacturing Process (atlocal CMO, controlled room and humidity conditions throughout theprocess):

Amount Ingredients (%) Uncoated Antibiotic Granules/Pellets 45%Excipients (Microcrystalline cellulose - filler, 28%polyvinylpyrrolidone - binder, pregelatinized starch - disintegrant andsilicon dioxide - flow aid, magnesium stearate - lubricant) pH 5.0 to6.0 Enteric Coated (EC) Antibiotic Granules/Pellets 13% pH 7.2 to 7.5Enteric Coated (EC) Symbiotic Granules/Pellets 13% HPMC or equivalent“polymers” (Film coat) 1% Water/Solvents as required 0%

The above Uncoated Antibiotic Granules/Pellets intermediate formulationsis blended in desired portions in V-type or similar blender withexcipients to aid in flow, disintegration and lubrication (for tabletingmachine). The blended powders are compressed into tablets usingtri-layer tableting equipment (“EC Tablets-1”). The above pH 5.0 to 6.0Enteric Coated (EC) Antibiotic Granules/Pellets intermediate formulationis blended in desired portions in V-type or similar blender withadditional excipients to aid in flow, disintegration and lubrication(for tableting machine). The blend is compressed over the first layer oftablets (EC Tablets-1) using tri-layer tableting equipment (“ECTablets-2”). The pH 7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets intermediate formulations are blended in desiredportions in V-type or similar blender with additional excipients to aidin flow, disintegration and lubrication (for tableting machine). Theblended powder is compressed over the second layer of tablets (ECtablets-2) using tri-layer tableting machine. The tablets are furtherfilm coated using aqueous or solvent coating solution in a coating panor fluid bed dryer using HPMC or equivalent “polymers” (Film coat).

Final Product Packaging (at local CMO, dry low humidity and low oxygen(N2 purging) conditions throughout the process):

The above granules are packaged in sachet, and the coated tablets,capsules are packaged into bottles with induction sealing or blisteredat low humidity (at or below 40% RH) and controlled room temperatureconditions (at 20 to 25 degrees C.).

Quality Control Release Testing (Active Pharmaceutical Ingredient (API)and Final Drug Product) Symbiotic—

Test Methods and Assessment Description Granules, pellets, tablets,capsules in blisters or bottles or sachets Appearance Visual inspectionfor color, shape, etc. Identification Genes, species, strains.Morphological appearance via Microscopic evaluation and/or multiplex PCRas well as other tests including biochemical methods such asfermentation profile or genotypic methods, e.g. ribotyping, restrictionfragment length polymorphism (RFLP), or both. In addition, develop aspecific identity assay for critical biological activity. Others testmay include: DNA-DNA hybridization to specify strains in species; DNAsequence coding per WHO; Strain typing include Pulsed Field Gelelectrophoresis (PFGE), etc. Potency - Viable Microscopic testing, orOpacity to measure viable cells per organisms unit or dose, i.e. colonyforming units (CFU) Potency Assay Assessment of CFU (on solid medium)and tests to correlating with activity. M-viability plating. PurityEndotoxin content, residual antibiotics, and/or the quantification ofresidual toxic components or contaminants introduced during manufactureby Elisa or amino acid profile Microbial bioburden or Extraneousmaterials including pathogens by using Elisa or contaminants and limitsamino acid profile or SDS page or ion exchange chromatography, etc.Microbial limits by US Pharmacopeia (USP 31 <61>). Percent viable cellsMicro testing after regrown in appropriate media and test, e.g.,Dead/live assay by ATP. Also determination of non-viable units per gi.e., by electro-zone count of non-fluorescent cells (SDS PAGE)Particulate matter USP 31 <788> Pyrogens TBD pH Testing pH meterResidual moisture Water content, USP 31 <921> Content Uniformity TBDPackage Integrity Leaker test by vacuum Stability Potency, viable celldetermination, microbial contamination, pH an residual moisture

Antibiotic(s)

Test Methods and Assessment Identification HPLC and other Assay HPLC andother Impurities and Related sub HPLC and other Content uniformity HPLCand other

Symbiotic and Antibiotic

Test Methods and Assessment In-vitro release testing Medium: pH 1 buffer(simulated gastric), (via dissolution testing pH 6 buffer, pH 7.2 to 7.5buffer (simulated equipment): USP intestinal fluid), followed by pH5.5-6.2 buffer paddle or basket (simulated colonic fluid). Sample Times:pH 1 buffer - 1 hour pH 6 buffer - 1 hour pH 7.2 to 7.5 - 1, 2, 3 and 4hours pH 5.5 to 6.2 - 1, 2, 4 and 8 hours Symbiotic Assay: Microbiologytesting for count (cfu/gram) for Antibiotic Assay: HPLC Stabilitytesting (0, 6, Symbiotic: 12, 18 and 24 months): Identification,Potency, viable cell determination, microbial contamination, pH andresidual moisture, etc. Antibiotic: Identification, Assay, Impurities,Related Substances, microbial contamination, pH and residual moisture,etc.

Fecal Microbiota Transplantation (MET).

Materials and Methods:

Described below are formulations that are being made and tested for thetarget delivery for testing in biological assays, the formulation havingan Healthy human bacterial fecal flora for release at pH 5.5-6.2 inright colon every 24 hours.

Active Pharmaceutical Ingredient (API):

-   -   Human bacterial fecal flora donated by health human volunteers,        screened for safety.    -   Osmotic agents: proteins (casein, hydrolyzed protein, etc.),        peptides, amino acids (L-Leucine), carbohydrates glucose,        lactose, starches, inulin, sodium chloride, phosphate buffers,        etc. Lallemand and other high quality global suppliers.    -   Inactive Ingredients (Excipients):    -   Fillers and carriers: Microcrystalline, starch, HPMC or        equivalent “polymers”, hard HPMC capsules, soft gelatin and        other materials, etc.—purchased from local US supplier such as        FMC, Capsugel, Colorcon, as well as pregelatinized        starch—disintegrant, silicon dioxide—flow aid, magnesium        stearate—lubricant) from various reputable excipient suppliers.

Intermediate Formulation/Manufacturing Process (at local CMO): “DriedHealthy Human Bacterial Fecal Flora”:

Amount Ingredients (%) Healthy human donor's bacterial fecal flora 40%Inactive ingredients - L. Leucine, sodium chloride, and/or 40% dextrose,etc. Inactive ingredients - phosphate buffer, tylexopol, and/or 20%sodium glutamate, etc. Water as required 0%

Dissolve the phosphate buffer, sodium chloride, and/or dextrose, etc. inwater. Add the healthy human donor's bacterial fecal flora material tothe mix and stir in a mixer. Pass the suspension through a large meshfilter to remove insoluble material (flora mix). Dissolve the phosphatebuffer, tylexopol, and/or sodium glutamate, etc. in water and the dilutethe flora mix. Fill into vials and freeze dry the mix or pass throughsprayer drier or foam drier to remove moisture and produce fine powder.

“Dried Human Bacterial Fecal Flora Granules” (75-100 micron range):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 25% Excipients(Microcrystalline cellulose - filler, 75% pregelatinized starch -disintegrant, silicon dioxide - flow aid, magnesium stearate -lubricant)

Prepare a dry granulation with Dried Human Bacterial Fecal Flora andexcipients in a low shear mixer.

Example

Final Product—Capsules (HPMC)—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 15% Polymers”(pH 5.5 to 6.2 sensitive coating) 25% Polymers” (pH 7.2 to 7.5 sensitivecoating) 25% HPMC or equivalent “polymers” (Barrier and Seal 5% coats)HPMC Capsules 30%

The above Dried Human Bacterial Fecal Flora is filled into smallcapsules using encapsulation equipment. The capsules are coated with pH5.5 to 6.2 sensitive coating using aqueous or solvent coating solutionof “Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are barrier coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Capsules (HPMC)—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Amount Ingredients (%) Dried Human Bacterial Fecal Flora Granules 68%Polymers” (pH 5.5 to 6.2 sensitive coating) 10% Polymers” (pH 7.2 to 7.5sensitive coating) 10% HPMC or equivalent “polymers” (Barrier and Sealcoats) 2% HPMC Capsules 10%

The above Dried Human Bacterial Fecal Flora Granules are filled intosmall capsules using encapsulation equipment. The capsules are coatedwith pH 5.5 to 6.2 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions. The coated capsules are barrier coated withaqueous or solvent coating solution of HPMC or equivalent “polymers” ina coating pan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Liquid Filled Soft Gelatin/Veggie GelCapsules—Formulation/Manufacturing Process (at local CMO, controlledroom temperature, humidity and oxygen conditions throughout theprocess):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 15% Vegetableoil (immiscible liquid) and/or other no-aqueous 53% ingredients (paste)Polymers” (pH 5.5 to 6.2 sensitive coating) 10% Polymers” (pH 7.2 to 7.5sensitive coating) 10% HPMC or equivalent “polymers” (Barrier and Sealcoats) 2% Vegetable gel mix or gelatin for producing veggie or soft 10%gelatin capsules

The above Dried Human Bacterial Fecal Flora is mixed with Vegetable oil(immiscible liquid) and/or other no-aqueous ingredients (paste) in ablender using optimum conditions. The mixture is filled with vegetablegel mix or gelatin in an encapsulation equipment for producing veggie orsoft gelatin capsules. The capsules are coated with pH 5.5 to 6.2sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are barrier coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Liquid Filled Hard Capsules (e.g.HPMC)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 15% Vegetableoil (immiscible liquid) and/or other no-aqueous 53% ingredients (paste)Polymers” (pH 5.5 to 6.2 sensitive coating) 10% Polymers” (pH 7.2 to 7.5sensitive coating) 10% HPMC or equivalent “polymers” (Barrier and Sealcoats) 2% Hard Gelatin/HPMC capsules 10%

The above Dried Human Bacterial Fecal Flora is mixed with Vegetable oil(immiscible liquid) and/or other no-aqueous ingredients (paste) in ablender using optimum conditions. The mixture is filled into hard HPMCcapsules using an encapsulation equipment. The capsules are coated withpH 5.5 to 6.2 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions. The coated capsules are barrier coated withaqueous or solvent coating solution of HPMC or equivalent “polymers” ina coating pan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Capsule-in-Capsule (HPMC)(1)—Formulation/ManufacturingProcess (at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora Granules 35%Excipients (Microcrystalline cellulose - filler, pregelatinized 33%starch - disintegrant, silicon dioxide - flow aid, magnesium stearate -lubricant) Polymers” (pH 5.5 to 6.2 sensitive coating) 10% Polymers” (pH7.2 to 7.5 sensitive coating) 10% HPMC or equivalent “polymers” (Barrierand Seal coats) 2% HPMC Capsules 10%

The above Dried Human Bacterial Fecal Flora Granules are filled intosmall capsules using encapsulation equipment. The capsules are coatedwith pH 5.5 to 6.2 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions. The coated capsules are barrier coated withaqueous or solvent coating solution of HPMC or equivalent “polymers” ina coating pan or fluid bed drier/coater using optimized conditions. Theabove Excipients along with the smaller filled capsules are furtherfilled into larger capsules using specialized capsule filling equipmentand optimized conditions. The larger capsules are further coated with pH7.2 to 7.5 sensitive coating using aqueous or solvent coating solutionof “Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are finally seal coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions.

Example

Final Product—Softgel Capsule-in-Capsule (e.g. softgelatin)(2)—Formulation/Manufacturing Process (at local CMO, controlledroom temperature, humidity and oxygen conditions throughout theprocess):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 15% Vegetableoil (immiscible liquid) and/or other 53% no-aqueous ingredients (paste)Polymers” (pH 5.5 to 6.2 sensitive coating) 10% Polymers” (pH 7.2 to 7.5sensitive coating) 10% HPMC or equivalent “polymers” (Barrier and Sealcoats) 2% Vegetable gel mix or gelatin for producing veggie or 10% softgelatin capsules

The above Dried Human Bacterial Fecal Flora is filled with vegetable gelmix or gelatin in encapsulation equipment for producing veggie or softgelatin capsules using optimum conditions. The veggie or soft gelatincapsules along with vegetable oil are together encapsulated usinganother encapsulation equipment for producing larger veggie or softgelatin capsules. The larger capsules are coated with pH 5.5 to 6.2sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are barrier coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Tablet-in-Capsule (HPMC)—Formulation/Manufacturing Process(at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora Granules 68%Polymers” (pH 5.5 to 6.2 sensitive coating) 10% Polymers” (pH 7.2 to 7.5sensitive coating) 10% HPMC or equivalent “polymers” (Barrier and Sealcoats) 2% HPMC Capsules 10%

The above Dried Human Bacterial Fecal Flora Granules are compressed intosoft microtablets using compression machine and optimum conditions. Themicrotablets are then filled into small capsules using encapsulationequipment. The capsules are coated with pH 5.5 to 6.2 sensitive coatingusing aqueous or solvent coating solution of “Polymers” in a coating panor fluid bed drier/coater using optimized conditions. The coatedcapsules are barrier coated with aqueous or solvent coating solution ofHPMC or equivalent “polymers” in a coating pan or fluid bed drier/coaterusing optimized conditions. The film-coated capsules are further coatedwith pH 7.2 to 7.5 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions. The coated capsules are finally seal coated withaqueous or solvent coating solution of HPMC or equivalent “polymers” ina coating pan or fluid bed drier/coater using optimized conditions.

Example

Final Product—Tablet-in-Capsule (Liquid Filled Soft Gelatin/VeggieGel)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora Granules 35%Vegetable oil (immiscible liquid) and/or other no-aqueous 33%ingredients (paste) Polymers” (pH 5.5 to 6.2 sensitive coating) 10%Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent“polymers” (Barrier and Seal coats) 2% Vegetable gel mix or gelatin forproducing veggie or 10% soft gelatin capsules

The above Dried Human Bacterial Fecal Flora Granules are compressed intosoft microtablets using compression machine and optimum conditions. Themicrotablets and the vegetable oil mix are filled with vegetable gel mixor gelatin in an encapsulation equipment for producing veggie or softgelatin capsules. The capsules are coated with pH 5.5 to 6.2 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are barrier coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The film-coated capsules arefurther coated with pH 7.2 to 7.5 sensitive coating using aqueous orsolvent coating solution of “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The coated capsules are finallyseal coated with aqueous or solvent coating solution of HPMC orequivalent “polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions.

Final Product Packaging (at local CMO, dry low humidity and low oxygen(N2 purging) conditions throughout the process). The above granules arepackaged in sachet, and the coated tablets, capsules are packaged intobottles with induction sealing or blistered at low humidity (at or below40% RH) and controlled room temperature conditions (at 20 to 25 degreesC.).

Quality Control Release Testing (Active Pharmaceutical Ingredient (API)and Final Drug Product)

Human Bacterial Fecal Flora

Test Methods and Assessment Description Powder, Granules, capsules inblisters or bottles or sachets Appearance Visual inspection for color,shape, etc. Identification Genes, species, strains. Morphologicalappearance via Microscopic evaluation and/or multiplex PCR as well asother tests including biochemical methods such as fermentation profileor genotypic methods, e.g. ribotyping, restriction fragment lengthpolymorphism (RFLP), or both. In addition, develop a specific identityassay for critical biological activity. Others test may include: DNA-DNAhybridization to specify strains in species; DNA sequence coding perWHO; Strain typing include Pulsed Field Gel electrophoresis (PFGE), etc.Potency - Viable Microscopic testing, or Opacity to measure viable cellsper organisms unit or dose, i.e. colony forming units (CFU) PotencyAssay Assessment of CFU (on solid medium) and tests to correlating withactivity. M-viability plating. Elisa or amino acid profile.Purity/Related Endotoxin content, antibiotic residue and/or thequantification substances of residual toxic components or contaminantsintroduced during manufacture by Elisa or amino acid profile; SDS pageand or amino acid profile. Microbial bioburden or Extraneous materialsincluding pathogens by using Elisa or contaminants and limits amino acidprofile or SDS page or ion exchange (related substances) chromatography,etc. Microbial limits by US Pharmacopeia (USP 31 <61>). Percent viablecells Micro testing after regrown in appropriate media and test, e.g.,Dead/live assay by ATP. Also determination of non-viable units per gi.e., by electro-zone count of non-fluorescent cells (SDS PAGE)Particulate matter USP 31 <788> Pyrogens TBD pH Testing pH meterResidual moisture Water content, USP 31 <921> Content Uniformity ATPLive/Dead Assay ATP Heavy metals Inductively Coupled Plasma-AtomicEmission Spectrophotometry (ICP-AES); Inductively Coupled Plasma- MassSpectroscopy (ICP-MS); Atomic Emission Spectrophotometry (AES); orAtomic Absorption Spectrophotometry (AAS). Water content Karl FischerPackage Integrity Leaker test by vacuum Stability Potency, viable celldetermination, microbial contamination, pH an residual moisture In-vitrorelease testing USP paddle or basket (via dissolution testing Medium: pH1 buffer (simulated gastric), pH 6 buffer, pH 7.2 equipment): to 7.5buffer (simulated intestinal fluid), followed by pH 5.5-6.2 buffer(simulated colonic fluid). Sample Times: pH 1 buffer - 1 hour pH 5.5-6.2buffer - 1, 2, 3 and 4 hours pH 7.2 to 7.5-1, 2, 3 and 4 hours pH 5.5 to6.2-1, 2, 4 and 8 hours Human Bacterial Fecal Flora - Assay:Microbiology testing for count (cfu/gram) Stability testing (0, 6,Identification, Appearance, Potency, viable cell determination, 12, 18and 24 months): microbial contamination, pH and residual moisture,related substance, water content, Live/dead Assay, etc.

Fecal Microbiota Transplantation (MET) with C. difficile anti-toxin(CDAT)

Materials and Methods:

Described below are formulations that are being made and tested for thetarget delivery for testing in biological assays, the formulation havingan Healthy human bacterial fecal flora for release at pH 5.5-6.2 inright colon every 24 hours.

Active Pharmaceutical Ingredient (API):

-   -   Human bacterial fecal flora donated by health human volunteers,        screened for safety.    -   C. difficile anti-toxin (CDAT) provided from specialty supplier    -   Osmotic agents: proteins (casein, hydrolyzed protein, etc.),        peptides, amino acids (L-Leucine), carbohydrates glucose,        lactose, starches, inulin, sodium chloride, phosphate buffers,        etc. Lallemand and other high quality global suppliers.    -   Inactive Ingredients (Excipients):    -   Fillers and carriers: Microcrystalline, starch, HPMC or        equivalent “polymers”, hard HPMC capsules, soft gelatin and        other materials, etc.—purchased from local US supplier such as        FMC, Capsugel, Colorcon, as well as pregelatinized        starch—disintegrant, silicon dioxide—flow aid, magnesium        stearate—lubricant) from various reputable excipient suppliers.

Intermediate Formulation/Manufacturing Process (at local CMO): “DriedHealthy Human Bacterial Fecal Flora”:

Ingredients Amount (%) Healthy human donor's bacterial fecal flora 40%Inactive ingredients - L. Leucine, sodium chloride, and/or 40% dextrose,etc. Inactive ingredients - phosphate buffer, tylexopol, and/or 20%sodium glutamate, etc. Water as required 0%

Dissolve the phosphate buffer, sodium chloride, and/or dextrose, etc. inwater. Add the healthy human donor's bacterial fecal flora material tothe mix and stir in a mixer. Pass the suspension through a large meshfilter to remove insoluble material (flora mix). Dissolve the phosphatebuffer, tylexopol, and/or sodium glutamate, etc. in water and the dilutethe flora mix. Fill into vials and freeze dry the mix or pass throughsprayer drier or foam drier to remove moisture and produce fine powder.

“Dried Human Bacterial Fecal Flora Granules” (75-100 micron range):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 25% Excipients(Microcrystalline cellulose - filler, pregelatinized 75% starch -disintegrant, silicon dioxide - flow aid, magnesium stearate -lubricant)

Prepare a dry granulation with Dried Human Bacterial Fecal Flora andexcipients in a low shear mixer. “CDAT Granules” (75-100 micron range):

Ingredients Amount (%) CDAT 25% Excipients (Microcrystalline cellulose -filler, pregelatinized 75% starch - disintegrant, silicon dioxide - flowaid, magnesium stearate - lubricant)

Prepare a dry granulation with CDAT and excipients in a low shear mixer.

Example

Final Product—Capsules (HPMC)—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 10% CDAT 5%Polymers” (pH 5.5 to 6.2 sensitive coating) 25% Polymers” (pH 7.2 to 7.5sensitive coating) 25% HPMC or equivalent “polymers” (Barrier and Sealcoats) 5% HPMC Capsules 30%

The above Dried Human Bacterial Fecal Flora and the CDAT is filled intosmall capsules using encapsulation equipment. The capsules are coatedwith pH 5.5 to 6.2 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions. The coated capsules are barrier coated withaqueous or solvent coating solution of HPMC or equivalent “polymers” ina coating pan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Capsules (HPMC)—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora Granules 34%CDAT Granules 34% Polymers” (pH 5.5 to 6.2 sensitive coating) 10%Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent“polymers” (Barrier and Seal coats) 2% HPMC Capsules 10%

The above Dried Human Bacterial Fecal Flora and CDAT Granules are filledinto small capsules using encapsulation equipment. The capsules arecoated with pH 5.5 to 6.2 sensitive coating using aqueous or solventcoating solution of “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The coated capsules are barriercoated with aqueous or solvent coating solution of HPMC or equivalent“polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The film-coated capsules are further coated with pH 7.2 to7.5 sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are finally seal coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions.

Example

Final Product—Liquid Filled Soft Gelatin/Veggie GelCapsules—Formulation/Manufacturing Process (at local CMO, controlledroom temperature, humidity and oxygen conditions throughout theprocess):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 10% CDAT 5%Vegetable oil (immiscible liquid) and/or other no-aqueous 53%ingredients (paste) Polymers” (pH 5.5 to 6.2 sensitive coating) 10%Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent“polymers” (Barrier and Seal coats) 2% Vegetable gel mix or gelatin forproducing veggie 10% or soft gelatin capsules

The above Dried Human Bacterial Fecal Flora and CDAT are mixed withVegetable oil (immiscible liquid) and/or other no-aqueous ingredients(paste) in a blender using optimum conditions. The mixture is filledwith vegetable gel mix or gelatin in encapsulation equipment forproducing veggie or soft gelatin capsules. The capsules are coated withpH 5.5 to 6.2 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions. The coated capsules are barrier coated withaqueous or solvent coating solution of HPMC or equivalent “polymers” ina coating pan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Liquid Filled Hard Capsules (e.g.HPMC)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 10% CDAT 5%Vegetable oil (immiscible liquid) and/or other no-aqueous 53%ingredients (paste) Polymers” (pH 5.5 to 6.2 sensitive coating) 10%Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent“polymers” (Barrier and Seal coats) 2% Hard Gelatin/HPMC capsules 10%

The above Dried Human Bacterial Fecal Flora and CDAT are mixed withVegetable oil (immiscible liquid) and/or other no-aqueous ingredients(paste) in a blender using optimum conditions. The mixture is filledinto hard HPMC capsules using encapsulation equipment. The capsules arecoated with pH 5.5 to 6.2 sensitive coating using aqueous or solventcoating solution of “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The coated capsules are barriercoated with aqueous or solvent coating solution of HPMC or equivalent“polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The film-coated capsules are further coated with pH 7.2 to7.5 sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are finally seal coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions.

Example

Final Product—Capsule-in-Capsule (HPMC)(1)—Formulation/ManufacturingProcess (at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Amount Ingredients (%) Dried Human Bacterial Fecal Flora Granules 23%CDAT Granules 12% Excipients (Microcrystalline cellulose - filler,pregelatinized 33% starch - disintegrant, silicon dioxide - flow aid,magnesium stearate - lubricant) Polymers” (pH 5.5 to 6.2 sensitivecoating) 10% Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC orequivalent “polymers” (Barrier and Seal coats) 2% HPMC Capsules 10%

The above Dried Human Bacterial Fecal Flora and CDAT Granules are filledinto small capsules using encapsulation equipment. The capsules arecoated with pH 5.5 to 6.2 sensitive coating using aqueous or solventcoating solution of “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The coated capsules are barriercoated with aqueous or solvent coating solution of HPMC or equivalent“polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The above Excipients along with the smaller filled capsulesare further filled into larger capsules using specialized capsulefilling equipment and optimized conditions. The larger capsules arefurther coated with pH 7.2 to 7.5 sensitive coating using aqueous orsolvent coating solution of “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The coated capsules are finallyseal coated with aqueous or solvent coating solution of HPMC orequivalent “polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions.

Example

Final Product—Capsule-in-Capsule (HPMC)(2)—Formulation/ManufacturingProcess (at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora Granules 23%CDAT Granules 12% Excipients (Microcrystalline cellulose - filler,pregelatinized 33% starch - disintegrant, silicon dioxide - flow aid,magnesium stearate - lubricant) Polymers” (pH 5.5 to 6.2 sensitivecoating) 10% Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC orequivalent “polymers” (Barrier and Seal coats) 2% HPMC Capsules 10%

The above Dried Human Bacterial Fecal Flora are filled into smallcapsules using encapsulation equipment. The capsules are coated with pH5.5 to 6.2 sensitive coating using aqueous or solvent coating solutionof “Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are barrier coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions. The aboveExcipients along with the, CDAT and smaller filled capsules are furtherfilled into larger capsules using specialized capsule filling equipmentand optimized conditions. The larger capsules are further coated with pH7.2 to 7.5 sensitive coating using aqueous or solvent coating solutionof “Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are finally seal coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions.

Example

Final Product—Softgel Capsule-in-Capsule (e.g. softgelatin)(3)—Formulation/Manufacturing Process (at local CMO, controlledroom temperature, humidity and oxygen conditions throughout theprocess):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora 10% CDAT 5%Vegetable oil (immiscible liquid) and/or other no-aqueous 53%ingredients (paste) Polymers” (pH 5.5 to 6.2 sensitive coating) 10%Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent“polymers” (Barrier and Seal coats) 2% Vegetable gel mix or gelatin forproducing veggie or 10% soft gelatin capsules

The above Dried Human Bacterial Fecal Flora is filled with vegetable gelmix or gelatin in encapsulation equipment for producing veggie or softgelatin capsules using optimum conditions. The veggie or soft gelatincapsules along with vegetable oil are together encapsulated usinganother encapsulation equipment for producing larger veggie or softgelatin capsules. The larger capsules are coated with pH 5.5 to 6.2sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are barrier coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Tablet-in-Capsule (HPMC)—Formulation/Manufacturing Process(at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora Granules45.3%   CDAT Granules 22.7%   Polymers” (pH 5.5 to 6.2 sensitivecoating) 10% Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC orequivalent “polymers” (Barrier and Seal coats)  2% HPMC Capsules 10%

The above Dried Human Bacterial Fecal Flora Granules are compressed intosoft microtablets using compression machine and optimum conditions. Themicrotablets and the CDAT Granules are then filled into small capsulesusing encapsulation equipment. The capsules are coated with pH 5.5 to6.2 sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are barrier coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Tablet-in-Capsule (Liquid Filled Soft Gelatin/VeggieGel)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) Dried Human Bacterial Fecal Flora Granules 23%CDAT 12% Vegetable oil (immiscible liquid) and/or other 33% no-aqueousingredients (paste) Polymers” (pH 5.5 to 6.2 sensitive coating) 10%Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent“polymers” (Barrier and Seal coats) 2% Vegetable gel mix or gelatin forproducing veggie or 10% soft gelatin capsules

The above Dried Human Bacterial Fecal Flora Granules are compressed intosoft microtablets using compression machine and optimum conditions. Themicrotablets, CDAT and the vegetable oil mix are filled with vegetablegel mix or gelatin in encapsulation equipment for producing veggie orsoft gelatin capsules. The capsules are coated with pH 5.5 to 6.2sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater using optimizedconditions. The coated capsules are barrier coated with aqueous orsolvent coating solution of HPMC or equivalent “polymers” in a coatingpan or fluid bed drier/coater using optimized conditions. Thefilm-coated capsules are further coated with pH 7.2 to 7.5 sensitivecoating using aqueous or solvent coating solution of “Polymers” in acoating pan or fluid bed drier/coater using optimized conditions. Thecoated capsules are finally seal coated with aqueous or solvent coatingsolution of HPMC or equivalent “polymers” in a coating pan or fluid beddrier/coater using optimized conditions.

Final Product Packaging (at local CMO, dry low humidity and low oxygen(N2 purging) conditions throughout the process). The above granules arepackaged in sachet, and the coated tablets, capsules are packaged intobottles with induction sealing or blistered at low humidity (at or below40% RH) and controlled room temperature conditions (at 20 to 25 degreesC.).

Quality Control Release Testing (Active Pharmaceutical Ingredient (API)and Final Drug Product). Human Bacterial Fecal Flora—

Test Methods and Assessment Description Bacterial flora and CDAT:Powder, Granules, capsules in blisters or bottles or sachets AppearanceBacterial flora and CDAT: Visual inspection for color, shape, etc.Identification Bacterial flora: Genes, species, strains. Morphologicalappearance via Microscopic evaluation and /or multiplex PCR as well asother tests including biochemical methods such as fermentation profileor genotypic methods, e.g. ribotyping, restriction fragment lengthpolymorphism (RFLP), or both. In addition, develop a specific identityassay for critical biological activity. Others test may include: DNA-DNAhybridization to specify strains in species; DNA sequence coding perWHO; Strain typing include Pulsed Field Gel electrophoresis (PFGE), etc.CDAT: Amino acid profile Potency Bacterial flora (Viable organisms):Microscopic testing, or Opacity to measure viable cells per unit ordose, i.e. colony forming units (CFU) CDAT: Elisa and amino acid profilePotency Assay Bacterial flora: Assessment of CFU (on solid medium) andtests to correlating with activity. M-viability plating. Elisa or aminoacid profile. CDAT: Elisa and amino acid profile Purity/RelatedBacterial flora: Endotoxin content, antibiotic residue and/or substancesthe quantification of residual toxic components or contaminantsintroduced during manufacture by Elisa or amino acid profile; SDS pageand or amino acid profile. CDAT: Elisa or amino acid profile; SDS pageand or amino acid profile. Microbial bioburden or Bacterial flora andCDAT: Extraneous materials including contaminants and limits pathogensby using Elisa or amino acid profile or SDS page or (related substances)ion exchange chromatography, etc. Microbial limits by US Pharmacopeia(USP 31 <61>). Percent viable cells Bacterial flora: Micro testing afterregrown in appropriate media and test, e.g., Dead/live assay by ATP.Also determination of non-viable units per g i.e., by electro-zone countof non-fluorescent cells (SDS PAGE) Particulate matter Bacterial floraand CDAT: USP 31 <788> Pyrogens Bacterial flora and CDAT: TBD pH TestingBacterial flora and CDAT: pH meter Residual moisture Bacterial flora andCDAT: Water content, USP 31 <921> Content Uniformity Bacterial flora:ATP CDAT: Elisa or amino acid profile Live/Dead Assay Bacterial flora:ATP Heavy metals Bacterial flora and CDAT: Inductively Coupled Plasma-Atomic Emission Spectrophotometry (ICP-AES); Inductively CoupledPlasma-Mass Spectroscopy (ICP-MS); Atomic Emission Spectrophotometry(AES); or Atomic Absorption Spectrophotometry (AAS). Water contentBacterial flora and CDAT: Karl Fischer Package Integrity Bacterial floraand CDAT: Leaker test by vacuum Stability Bacterial flora: Potency,viable cell determination, microbial contamination, pH an residualmoisture CDAT: Potency, pH an residual moisture In-vitro release testingBacterial flora: USP paddle or basket (via dissolution testing Medium:pH 1 buffer (simulated gastric), pH 6 buffer, pH 7.2 equipment): to 7.5buffer (simulated intestinal fluid), followed by pH 5.5-6.2 buffer(simulated colonic fluid). Sample Times: pH 1 buffer - 1 hour pH 5.5-6.2buffer - 1, 2, 3 and 4 hours pH 7.2 to 7.5 - 1, 2, 3 and 4 hours pH 5.5to 6.2 - 1, 2, 4 and 8 hours Human Bacterial Fecal Flora - Assay:Microbiology testing for count (cfu/gram); CDAT: Assay Stability testing(0, 6, Bacterial flora: Identification, Appearance, Potency, viable 12,18 and 24 months): cell determination, microbial contamination, pH andresidual moisture, related substance, water content, Live/dead Assay,etc. CDAT: Identification, Appearance, Potency, pH and residualmoisture, related substance, water content, etc.

Example 2: Obesity, Metabolic Syndrome and Type 2 Diabetes

Obesity results from alterations in the body's regulation of energyintake, expenditure, and storage. Animal and human data demonstrate thatphylogenic changes occur in the microbiota composition in obeseindividuals. Furthermore, evidence from animal models suggest that thealterations of the gut microbiota with obesity results in increasedenergy extraction and lipid deposition, altered release ofentero-hormones, increased intestinal permeability and metabolicendotoxemia. Treatment with pre- and probiotics may reverse many ofmetabolic effects linked with the altered microbiota in obese patients.The gut microbiota is, therefore, a potential nutritional andpharmacological target for the management of obesity and obesity-relateddisorders (12).

Materials and Methods:

Described below are methods and materials toward the making and testingof a formulation according to the invention for the treatment ofMetabolic Syndrome, Obesity and type 2 diabetes.

Target Delivery: Target Delivery: Symbiotic (prebiotic: L-Leucineprobiotic: live species of lactobacillus, bifidobacterium andFaecalibacterium prausnitzii) for release at 7.2-7.5 in ileum every 24hours.

Active Pharmaceutical Ingredient (API): Prebiotics—proteins (casein,hydrolyzed protein, etc.), peptides, amino acids (L-Leucine),carbohydrates glucose, lactose, starches, dextrose monohydrate, inulin,etc.: provided by Roquette, etc. and certain bacterial strains: providedby Denisco, CHR Hansen, Institu Risell—Lallemand and other high qualityglobal suppliers of prebiotics. Live probiotics Species of:lactobacillus, bifidobacterium and Faecalibacterium prausnitzii areprovided by Denisco, CHR Hansen, Institu Risell—Lallemand and other highquality global suppliers.

Inactive Ingredients (Excipients): Microcrystalline, pregelatinizedstarch, polyvinylpyrrolidone, silicon dioxide, HPMC or equivalent“polymers”, hard gelatin capsules, and other fillers, etc.—purchasedfrom local US supplier such as FMC, Capsugel, Colorcon, Evonik, etc.Intermediate Formulation/Manufacturing Process (at local CMO, controlledroom and humidity conditions throughout the process):

“Uncoated Symbiotic Granules/Pellets” (100 micron)

Ingredients Amount (%) Freeze dried bacteria (species of lactobacillus,0.90%   bifidobacterium and faecalibacterium prausnitzii) (probiotic)L-Leucine 0.1%  Excipients (Microcrystalline cellulose - filler, 99%polyvinylpyrrolidone - binder, pregelatinized starch - disintegrant,silicon dioxide - flow aid, magnesium stearate - lubricant) Water asrequired  0%

Prepared by mixing 1-leucine, suspension or freeze dried bacteria(species of lactobacillus, bifidobacterium and faecalibacteriumprausnitzii) with water and further spray/freeze dried to remove waterusing optimized conditions. The probiotic powder is mixed withexcipients in V-blender or similar blender.

“pH 7.2 to 7.5 Enteric Coated Symbiotic Granules/Pellets” (100 micron)

Ingredients Amount (%) Uncoated Symbiotic Granules/Pellets 95% HPMC orequivalent “polymers” (Barrier and Seal coats) 1% “Polymers” (pH 7.2 to7.5 sensitive coating) 4% Water/Solvents as required 0%

The Uncoated Symbiotic Granules/Pellets are coated (the barrier coat)with aqueous or solvent coating solution of HPMC or equivalent“polymers” to coat in a coating pan or fluid bed drier/coater usingoptimized conditions. The barrier coated micropellets or granules arefurther coated with aqueous or solvent coating solution of pH 7.2 to7.5, sensitive coating “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The above pH 7.2 to 7.5sensitive coated micropellets or granules are seal coated with aqueousor solvent coating solution of HPMC or equivalent “polymers” in acoating pan or fluid bed drier/coater using optimized conditions.

“Uncoated Dextrose Monohydrate Pellets/Granules”

Ingredients Amount (%) Dextrose monohydrate 80% Excipients(Microcrystalline cellulose - filler, 20% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant, silicon dioxide - flow aid,magnesium stearate - lubricant) Water as required 0%

Prepared by dry and/or wet granulating dextrose monohydrate, withexcipients in high or low shear mixer and further pelletizing usingextruder/spheronizer and then drying to remove water using optimizedconditions.

“pH 7.2 to 7.5 Enteric Coated Dextrose Monohydrate Granules/Pellets”(100 micron)

Ingredients Amount (%) Uncoated Dextrose Monohydrate Granules/Pellets95% HPMC or equivalent “polymers” (Barrier and Seal coats) 1% “Polymers”(pH 7.2 to 7.5 sensitive coating) 4% Water/Solvents as required 0%

The Uncoated Dextrose Monohydrate Granules/Pellets are further coated(barrier coat) with aqueous or solvent coating solution of HPMC orequivalent “polymers” in a coating pan or fluid bed drier/coater usingoptimized conditions. The above micropellets or granules are furthercoated with aqueous or solvent coating solution of “Polymers” (pH 7.2 to7.5 sensitive coating) in a coating pan or fluid bed drier/coater usingoptimized conditions. The above micropellets or granules are seal coatedwith aqueous or solvent coating solution of HPMC or equivalent“polymers” in a coating pan or fluid bed drier/coater using optimizedconditions.

Example

Final Product—Sachet—Formulation/Manufacturing Process (at local CMO,controlled room and humidity conditions throughout the process):

Intermediate Formulation Amount (%) pH 7.2 to 7.5 Enteric Coated (EC)Symbiotic 5% Granules/Pellets pH 7.2 to 7.5 Enteric Coated (EC) Dextrose90% Monohydrate Granules/Pellets Excipients (Mannitol - filler, Silicon5% dioxide - glidant/flow aid)

The above pH 7.2 to 7.5 Enteric Coated (EC) Symbiotic and DextroseMonohydrate Granules/Pellets intermediate formulations are blended indesired portions in V-type or similar blender with excipients to aid inflow. The blended powders are filled into sachets using powder fillingequipment.

Example

Final Product—Capsules (Hard gelatin/HPMC)—Formulation/ManufacturingProcess (at local CMO, controlled room and humidity conditionsthroughout the process):

Ingredients Amount (%) Uncoated Symbiotic Granules/Pellets 1% UncoatedDextrose Monohydrate Granules/Pellets 83% Excipients (Microcrystallinecellulose - filler, 7% Silicon dioxide - glidant/flow aid) HardGelatin/HPMC Capsules 7% “Polymers” (pH 7.2 to 7.5 sensitive coating) 2%Water/Solvents as required 0%

The above Uncoated Symbiotic and Dextrose Monohydrate Granules/Pelletsintermediate formulations are blended in desired portions in V-type orsimilar blender with excipients to aid in flow. The blended powders arefilled into capsules using encapsulating equipment. The filled capsulesare further coated with pH 7.2 to 7.5 sensitive coating using aqueous orsolvent coating solution of “Polymers” in a coating pan or fluid beddrier/coater with optimized conditions.

Example

Final Product—Capsules (Hard gelatin/HPMC)—Formulation/ManufacturingProcess (at local CMO, controlled room and humidity conditionsthroughout the process):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets 1% pH 7.2 to 7.5 Enteric Coated (EC) DextroseMonohydrate 85% Granules/Pellets Excipients (Microcrystallinecellulose - filler, Silicon dioxide - 7% glidant/flow aid) HardGelatin/HPMC Capsules 7%

The above pH 7.2 to 7.5 Enteric Coated (EC) Symbiotic and DextroseMonohydrate Granules/Pellets intermediate formulations are blended indesired portions in V-type or similar blender with excipients to aid inflow. The blended powders are filled into capsules using encapsulatingequipment

Example

Final Product—Capsules (Hard gelatin/HPMC)(2) Formulation/ManufacturingProcess (at local CMO, controlled room and humidity conditionsthroughout the process):

Ingredients Amount (%) Uncoated Symbiotic Granules/Pellets 1% UncoatedDextrose Monohydrate Granules/Pellets 81% Excipients (Microcrystallinecellulose - filler, 7% Silicon dioxide - glidant/flow aid) HardGelatin/HPMC Capsules 7% “Polymers” (pH 7.2 to 7.5 sensitive coating) 4%Water/Solvents as required 0%

The above Uncoated Symbiotic and Dextrose Monohydrate Granules/Pelletsintermediate formulations are blended in desired portions in V-type orsimilar blender with excipients to aid in flow. The blended powders arefilled into capsules using encapsulating equipment. The filled capsulesare enteric coated using aqueous or solvent coating solution ofPolymers” (pH 7.2 to 7.5 sensitive coating) in coating pan or fluid bedcoating equipment using optimized conditions.

Example

Final Product—Capsules Co-pack (2)(Hard Gelatin/HPMCcapsules)—Formulation/Manufacturing Process (at local CMO, controlledroom and humidity conditions throughout the process):

Ingredients Amount (%) Uncoated Symbiotic Granules/Pellets 1% UncoatedDextrose Monohydrate Granules/Pellets 81% Excipients (Microcrystallinecellulose - filler, 7% Silicon dioxide - glidant/flow aid) HardGelatin/HPMC Capsules 7% “Polymers” (pH 7.2 to 7.5 sensitive coating) 4%Water/Solvents as required 0%

The above Uncoated Symbiotic Granules/Pellets intermediate formulationis blended in desired portions in V-type or similar blender withexcipients. The blended powders are filled into smaller capsules usingencapsulating equipment. The filled capsules are enteric coated usingaqueous or solvent coating solution of Polymers” (pH 7.2 to 7.5sensitive coating) in coating pan or fluid bed coating equipment usingoptimized conditions. The above Uncoated Dextrose MonohydrateGranules/Pellets intermediate formulation is blended in desired portionsin V-type or similar blender with excipients to aid in flow. The blendedpowders are filled into capsules using encapsulating equipment. Thefilled capsules are enteric coated using aqueous or solvent coatingsolution of Polymers” (pH 7.2 to 7.5 sensitive coating) in coating panor fluid bed coating equipment using optimized conditions. The Twocapsules products are co-packed

Example

Final Product—Capsules-Capsules Co-pack (2)(Liquid Filled Hard or SoftGelatin/Hard Gelatin/HPMC capsules)—Formulation/Manufacturing Process(at local CMO, controlled room and humidity conditions throughout theprocess):

Amount Ingredients (%) Uncoated Coated (EC) Symbiotic Granules/Pellets1% Vegetable oil (immiscible liquid) 8.5%   Gelatin as powder and HardGelatin Capsules 0.5%   Uncoated Dextrose Monohydrate Granules/Pellets76%  Excipients (Microcrystalline cellulose - filler, Silicon dioxide -6% glidant/flow aid) Hard Gelatin/HPMC Capsules 6% “Polymers” (pH 7.2 to7.5 sensitive coating) 2% Water/Solvents as required 0%

The above Uncoated Symbiotic Granules/Pellets intermediate formulationis blended in desired portions with Vegetable oil (immiscible liquid) ina blender. Filled into capsules using soft or hard gelatin encapsulatingequipment. The filled capsules are enteric coated using aqueous orsolvent coating solution of Polymers” (pH 7.2 to 7.5 sensitive coating)in coating pan or fluid bed coating equipment using optimizedconditions.

The above Uncoated Dextrose Monohydrate Granules/Pellets is blended indesired portion with excipients in V-type or similar. The blender isfilled into capsules using encapsulating equipment. The filled capsulesare further coated with pH 7.2 to 7.5 sensitive coating using aqueous orsolvent coating solution of “Polymers” in a coating pan or fluid beddrier/coater with optimized conditions.

Example

Final Product—Tablets/Microtablets—Formulation/Manufacturing Process (atlocal CMO, controlled room and humidity conditions throughout theprocess):

Ingredients Amount (%) Uncoated Symbiotic Granules/Pellets 1% UncoatedDextrose Monohydrate Granules/Pellets 81% Excipients (Microcrystallinecellulose - filler, 13% polyvinylpyrrolidone, pregelatinized starch -disintegrant and silicon dioxide - flow aid, magnesium stearate -lubricant) HPMC or equivalent “polymers” (Barrier coat) 1% “Polymers”(pH 7.2 to 7.5 sensitive coating) 4% Water/Solvents as required 0%

The above Uncoated Symbiotic and Dextrose Monohydrate Granules/Pelletsintermediate formulations are blended in desired portions in V-type orsimilar blender with to aid in flow, disintegration and lubrication (fortableting machine). The blended powders are compressed intoTablets/Microtablets using tableting equipment. The tablets are furtherbarrier coated in a coating pan or fluid bed dryer using aqueous orsolvent coating solution of HPMC or equivalent “polymers” (Barriercoat). The barrier coated tablets are further enteric coated usingaqueous or solvent coating solution of Polymers” (pH 7.2 to 7.5sensitive coating) in coating pan or fluid bed coating equipment usingoptimized conditions.

Example

Final Product—Orally disintegrating Tablets(ODT)—Formulation/Manufacturing Process (at local CMO, controlled roomand humidity conditions throughout the process):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets 1% pH 7.2 to 7.5 Enteric Coated (EC) DextroseMonohydrate 85% Granules/Pellets Excipients (Microcrystallinecellulose - filler, pregelatinized 14% starch - disintegrant and silicondioxide - flow aid, magnesium stearate - lubricant)

The pH 7.2 to 7.5 Enteric Coated (EC) Symbiotic and Dextrose MonohydrateGranules/Pellets intermediate formulation are blended in desiredportions in V-type or similar blender with additional excipients to aidin flow. The blended powders are compressed into soft tablets usingtableting equipment.

Example

Final Product—Tablets Co-pack (2)(Hard Gelatin/HPMCcapsules)—Formulation/Manufacturing Process (at local CMO, controlledroom and humidity conditions throughout the process):

Ingredients Amount (%) Uncoated Symbiotic Granules/Pellets 1% UncoatedDextrose Monohydrate Granules/Pellets 81% Excipients (Microcrystallinecellulose - filler, Silicon 14% dioxide - glidant/flow aid) HPMC orequivalent “polymers” (Barrier coat) 1% “Polymers” (pH 7.2 to 7.5sensitive coating) 4% Water/Solvents as required 0%

The above Uncoated Symbiotic Granules/Pellets intermediate formulationis blended in desired portions in V-type or similar blender withexcipients. The blended powders are compressed into Tablets/Microtabletsusing tableting equipment. The tablets are further barrier coated in acoating pan or fluid bed dryer using aqueous or solvent coating solutionof HPMC or equivalent “polymers” (Barrier coat). The barrier coatedtablets are further enteric coated using aqueous or solvent coatingsolution of Polymers” (pH 7.2 to 7.5 sensitive coating) in coating panor fluid bed coating equipment using optimized conditions. The aboveUncoated Dextrose Monohydrate Granules/Pellets intermediate formulationis blended in desired portions in V-type or similar blender withexcipients. The blended powders are compressed into Tablets/Microtabletsusing tableting equipment. The tablets are further barrier coated in acoating pan or fluid bed dryer using aqueous or solvent coating solutionof HPMC or equivalent “polymers” (Barrier coat). The barrier coatedtablets are further enteric coated using aqueous or solvent coatingsolution of Polymers” (pH 7.2 to 7.5 sensitive coating) in coating panor fluid bed coating equipment using optimized conditions. The twotablet products are co-packed.

Final Product Packaging (at local CMO, dry low humidity and low oxygen(N2 purging) conditions throughout the process). The above granules arepackaged in sachet, and the coated tablets, as well as capsules arepackaged into bottles with induction sealing or blistered (co-packs) atlow humidity (at or below 40% RH) and controlled room temperatureconditions (at 20 to 25 degrees C.).

Quality Control Release Testing (Active Pharmaceutical Ingredient (API)and Final Drug Product)

Symbiotic—

Test Methods and Assessment Description Granules, pellets, tablets,capsules in blisters or bottles or sachets Appearance Visual inspectionfor color, shape, etc. Identification Genes, species, strains.Morphological appearance via Microscopic evaluation and/or multiplex PCRas well as other tests including biochemical methods such asfermentation profile or genotypic methods, e.g. ribotyping, restrictionfragment length polymorphism (RFLP), or both. In addition, develop aspecific identity assay for critical biological activity. Others testmay include: DNA-DNA hybridization to specify strains in species; DNAsequence coding per WHO; Strain typing include Pulsed Field Gelelectrophoresis (PFGE), etc. Potency - Viable Microscopic testing, orOpacity to measure viable cells per organisms unit or dose, i.e. colonyforming units (CFU) Potency Assay Assessment of CFU (on solid medium)and tests to correlating with activity. M-viability plating. PurityEndotoxin content, residual antibiotics, and/or the quantification ofresidual toxic components or contaminants introduced during manufactureby Elisa or amino acid profile Microbial bioburden or Extraneousmaterials including pathogens by using Elisa or contaminants and limitsamino acid profile or SDS page or ion exchange chromatography, etc.Microbial limits by US Pharmacopeia (USP 31 <61>). Percent viable cellsMicro testing after regrown in appropriate media and tests, e.g.,Dead/live assay by ATP. Also determination of non- viable units per gi.e., by electro-zone count of non-fluorescent cells (SDS PAGE)Particulate matter USP 31 <788> Pyrogens Rabbit pyrogencity test (USP 31<151>) pH Testing pH meter Residual moisture Water content, USP 31 <921>Content Uniformity ATP Package Integrity Leaker test by vacuum StabilityPotency, viable cell determination, microbial contamination, pH anresidual moisture In-vitro release testing Medium: pH 1 buffer(simulated gastric), pH 6 buffer, pH 7.2 (via dissolution testing to 7.5buffer (simulated intestinal fluid), followed by pH 5.5-6.2 equipment):USP buffer (simulated colonic fluid). paddle or basket Sample Times: pH1 buffer - 1 hour pH 6 buffer - 1 hour pH 7.2 to 7.5 - 1, 2, 3 and 4hours pH 7.2 to 7.5 - 1, 2, 4 and 8 hours Symbiotic Assay: Microbiologytesting for count (cfu/gram) for Stability testing (0, 6, Symbiotic: 12,18 and 24 months): Identification, Potency, viable cell determination,microbial contamination, pH and residual moisture, etc.

Example 3

Example 3 is directed toward the making and testing of a formulationaccording to the invention for the treatment of a Gastro intestinalreflux disease (GERD).

GERD is a chronic symptom of mucosal damage caused by stomach acidcoming up from the stomach into the esophagus. GERD is usually caused bychanges in the barrier between the stomach and the esophagus, includingabnormal relaxation of the lower esophageal sphincter, which normallyholds the top of the stomach closed, impaired expulsion of gastricreflux from the esophagus, or a hiatal hernia. These changes may bepermanent or temporary.

Treatment is typically via lifestyle changes and medications such asproton pump inhibitors, H2 receptor blockers or antacids with or withoutalginic acid. Surgery may be an option in those who do not improve. Inthe Western world between 10 and 20% of the population is affected.Probiotics or Fecal Microbiota For Transplation (FMT) (subject onanother patent application) may also help in balancing microbiota beforeand after usage of proton pump inhibitors.

Materials and Methods:

Described below are formulations that are being made and tested for thetarget delivery for testing in chemical and biological assays, theformulation having an proton pump inhibitor (e.g. Omeprazole magnesium,22.4 mg equivalent to 20 mg base (range: 10-40 mg)) (millimeter range)for release at pH 7.2-7.5 in ileum and symbiotic (prebiotic: L-Leucine;probiotic: species of: lactobacillus and bifidobacterium) to FMT forrelease at pH 5.5-6.2 in right colon every 24 hours.

Active Pharmaceutical Ingredient (API): Proton pump inhibitor—Forexample, omeprazole supplied by local generic US/non-US suppliers, e.g.,Manus Aktteva, etc. Prebiotics—proteins (casein, hydrolyzed protein,etc.), peptides, amino acids (L-Leucine), carbohydrates glucose,lactose, starches, inulin, etc. and certain bacterial strains: providedby Denisco, CHR Hansen, Institu Risell—Lallemand and other high qualityglobal suppliers of prebiotics. Live probiotics Species of:lactobacillus and bifidobacterium provided by Denisco, CHR Hansen,Institu Risell—Lallemand and other high quality global suppliers or FMTfrom volunteers.

Inactive Ingredients (Excipients): Microcrystalline, starch, HPMC orequivalent “polymers”, hard gelatin capsules, and other fillers,etc.—purchased from local US supplier such as FMC, Capsugel, Colorcon,as well as polyvinylpyrrolidone—binder, pregelatinizedstarch—disintegrant, silicon dioxide—flow aid, magnesiumstearate-lubricant) from various reputable excipient suppliers.

Intermediate Formulation/Manufacturing Process (at local CMO): “UncoatedProton pump inhibitor Granules/Pellets” (100 micron range):

Ingredients Amount (%) Proton pump inhibitor 13% Excipients(Microcrystalline cellulose - filler, 87% polyvinylpyrrolidone - binder,pregelatinized starch - disintegrant, silicon dioxide - flow aid,magnesium stearate - lubricant) Water as required 0%

Prepare a dry granulation with proton pump inhibitor and excipients in alow or high shear mixer and/or perform wet granulations withwater/solvent and further pelletize using extruder/spheronizer and thendrying to remove excess water/solvent using optimized conditions.

“pH 7.2 to 7.5 Enteric Coated (EC) Proton Pump InhibitorGranules/Pellets” (100 Micron Range):

Ingredients Amount (%) Uncoated Proton pump inhibitor Granules/Pellets88% HPMC or equivalent “polymers” (Barrier and Seal coats) 2% “Polymers”(pH 7.2 to 7.5 sensitive coating) 10% Water/Solvents as required 0%

The Uncoated Proton pump inhibitor Granules/Pellets are coated (thebarrier coat) with aqueous or solvent coating solution of HPMC orequivalent “polymers” to coat in a coating pan or fluid bed drier/coaterusing optimized conditions. The barrier coated micropellets or granulesare further coated with aqueous or solvent coating solution of pH 7.2 to7.5, sensitive coating “Polymers” in a coating pan or fluid beddrier/coater using optimized conditions. The above pH 7.2 to 7.5,sensitive coated micropellets or granules are seal coated with aqueousor solvent coating solution of HPMC or equivalent “polymers” in acoating pan or fluid bed drier/coater using optimized conditions.

“Uncoated Symbiotic Granules/Pellets” (100 Micron Range):

Ingredients Amount (%) L. Leucine (prebiotic) 5% Freeze dried bacteria(appropriate species and strains of 3% lactobacillus andbifidobacterium) probiotic) Excipients (Microcrystalline cellulose -filler, 92% polyvinylpyrrolidone - binder, pregelatinized starch -disintegrant, silicon dioxide - flow aid, magnesium stearate -lubricant)

Prepare a dry blend with prebiotic, freeze dried bacteria and excipientsin a low or high shear mixer.

“pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pellets” (100Micron).

Ingredients Amount (%) Uncoated Symbiotic Granules/Pellets 88% HPMC orequivalent “polymers” (Barrier and Seal coats) 2% “Polymers” (pH 5.5 to6.2 sensitive coating) 10% Water/Solvents as required 0%

The Uncoated Symbiotic Granules/Pellets are coated (barrier coat) withaqueous or solvent coating solution of HPMC or equivalent “polymers” ina coating pan or fluid bed drier/coater using optimized conditions. Theabove barrier coated micropellets or granules are further coated withaqueous or solvent coating solution of “Polymers” (pH 5.5 to 6.2sensitive coating) in a coating pan or fluid bed drier/coater usingoptimized conditions. The above pH 5.5 to 6.2 coated micropellets orgranules are seal coated with aqueous or solvent coating solution ofHPMC or equivalent “polymers” in a coating pan or fluid bed drier/coaterusing optimized conditions.

“pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets” (100 Micron).

Amount Ingredients (%) pH 5.5 to 6.2 Enteric Coated (EC) SymbioticGranules/Pellets 88% HPMC or equivalent “polymers” (Barrier and sealcoats) 2% “Polymers” (pH 7.2 to 7.5 sensitive coating) 10%Water/Solvents as required 0%

The above pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pelletsare coated with aqueous or solvent coating solution of “Polymers” (pH7.2 to 7.5 sensitive coating) in a coating pan or fluid bed drier/coaterusing optimized conditions. The above micropellets or granules arefurther coated with aqueous or solvent coating solution of HPMC orequivalent “polymers” (seal coat) in a coating pan or fluid beddrier/coater using optimized conditions.

Example

Final Product—Sachet—Formulation/Manufacturing Process (at local CMO,controlled room temperature, humidity and oxygen conditions throughoutthe process):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Mannitol - filler,Silicon dioxide - glidant/flow aid) 55%

The above pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets and pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions in V-type orsimilar blender with excipients using optimized conditions. The blendedpowders are filled into sachets using powder filling equipment.

Example

Final Product—Powder for Reconstitution—Formulation/ManufacturingProcess (at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Mannitol - filler,Silicon dioxide - glidant/flow aid) 55% Diluent 100 mL

The above pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets and pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions in V-type orsimilar blender with excipients using optimized conditions. The blendedpowders are filled into bottles (induction sealed) or pouches (sealed)using powder filling equipment.

Example

Final Product—Fast Dispersible Tablets—Formulation/Manufacturing Process(at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Mannitol - filler,Silicon dioxide - glidant/flow aid) 55% Diluent 100 mL

The above pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets and pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions in V-type orsimilar blender with excipients using optimized conditions. The blendedpowders are compressed to produce small tablets with scoring for ease ofdosing for pediatric applications.

Example

Final Product—Capsules (Hard gelatin/HPMC)—Formulation/ManufacturingProcess (at local CMO, controlled room temperature, humidity and oxygenconditions throughout the process):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Microcrystallinecellulose - filler, Silicon dioxide - 45% glidant/flow aid) HardGelatin/HPMC Capsules 10%

The above pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets and pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions in V-type orsimilar blender with excipients. The blended powders are filled intocapsules using encapsulating equipment.

Example

Final Product—Capsules (Liquid Filled Hard or SoftGelatin)—Formulation/Manufacturing Process (at local CMO, controlledroom temperature, humidity and oxygen conditions throughout theprocess):

Amount Ingredients (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Vegetable oil (immiscible liquid)and other ingredients (paste) 50% Gelatin as powder and Hard GelatinCapsules 5%

The above pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets and pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions withimmiscible liquid in a blender. Filled into capsules using soft or hardgelatin encapsulating equipment using optimized conditions.

Example

Final Product—Capsule-in-Capsule (Hard gelatin) (1)Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Amount Ingredients (%) pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic 15% Granules/Pellets pH 7.2 to 7.5 Enteric Coated (EC) Protonpump inhibitor 30% Granules/Pellets Excipients (Microcrystallinecellulose - filler, Silicon dioxide - 47% glidant/flow aid) Small andLarge Hard Gelatin/HPMC Capsules 8%

The pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets is blended in with portion of excipients in V-type orsimilar blender and the blend. The blend is filled into smaller capsulesusing encapsulating equipment and optimized conditions. The above pH 7.2to 7.5 Enteric Coated (EC) Proton pump inhibitor Granules/Pellets areblended together in desired portions in V-type or similar blender withexcipients. The blended intermediate formulations along with the smallerfilled capsules are further filled into larger capsules usingspecialized capsule filling equipment and optimized conditions.

Example

Final Product—Capsule-in-Capsule (Hard gelatin)(2)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Amount Ingredients (%) pH 5.5 to 6.2 Enteric Coated (EC) SymbioticGranules/Pellets 15% “Polymers” (pH 7.2 to 7.5 sensitive coating) 10%Excipients (Microcrystalline cellulose - filler, Silicon dioxide - 37%glidant/flow aid) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets Small and Large Hard Gelatin/HPMCCapsules 8% Water/Solvents as required 0%

The pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pellets isblended in desired portions in V-type or similar blender withexcipients. The blend is filled into smaller capsules usingencapsulating equipment. The smaller filled capsules are further coatedwith pH 7.2 to 7.5 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater withoptimized conditions. The above pH 7.2 to 7.5 Enteric Coated (EC) Protonpump inhibitor Granules/Pellets are blended in desired portions inV-type or similar blender with excipients. The smaller pH 7.2 to 7.5coated capsules and the blends are further filled into larger capsulesusing specialized capsule filling equipment and optimized conditions.

Example

Final Product—Capsule-in-Capsule (Hard gelatin) (3)Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Amount Ingredients (%) pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic 15% Granules/Pellets Uncoated Proton pump inhibitorGranules/Pellets 25% Excipients (Microcrystalline cellulose - filler,Silicon dioxide - 40% glidant/flow aid) “Polymers” (pH 7.2 to 7.5sensitive coating) 10% Small and Large Hard Gelatin/HPMC Capsules 10%

The above uncoated Proton pump inhibitor Granules/Pellets and a portionof excipients are blended together in V-type or similar blender. Theblend is filled into smaller capsules using encapsulating equipment andoptimized conditions. The smaller filled capsules are further coatedwith pH 7.2 to 7.5 sensitive coating using aqueous or solvent coatingsolution of “Polymers” in a coating pan or fluid bed drier/coater withoptimized conditions. The pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets is blended in with portion of excipients inV-type or similar blender. The blended intermediate formulations alongwith the smaller pH 7.2 to 7.5 EC capsules are further filled intolarger capsules using specialized capsule filling equipment andoptimized conditions.

Example

Final Product—Capsule-in-Capsule (Hard gelatin)(4)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Amount Ingredients (%) Uncoated Symbiotic Granules/Pellets 12% UncoatedProton pump inhibitor Granules/Pellets 30% Excipients (Microcrystallinecellulose - filler, Silicon dioxide - 30% glidant/flow aid) “Polymers”(pH 5.6 to 6.2 sensitive coating) 10% “Polymers” (pH 7.2 to 7.5sensitive coating) 10% Small and Large Hard Gelatin/HPMC Capsules 8%

The Uncoated Symbiotic Granules/Pellets is blended in with portion ofexcipients in V-type or similar blender. The blend is filled intosmaller capsules using encapsulating equipment and optimized conditions.The smaller filled capsules are further coated with pH 5.6 to 6.2sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater with optimizedconditions. The above uncoated Proton pump inhibitor Granules/Pelletsare blended together in desired portions in V-type or similar blenderwith excipients. The blended intermediate formulations along with thesmaller pH 5.6 to 6.2 EC capsules are further filled into largercapsules using specialized capsule filling equipment and optimizedconditions. The larger capsules are further coated with pH 7.2 to 7.5sensitive coating using aqueous or solvent coating solution of

“Polymers” in a coating pan or fluid bed drier/coater with optimizedconditions.

Example

Final Product—Orally disintegrating Tablets(ODT)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Mannitol - filler,polyvinylpyrrolidone - binder, 55% pregelatinized starch - disintegrantand silicon dioxide - flow aid, magnesium stearate - lubricant)

The Uncoated Proton pump inhibitor Granules/Pellets, pH 7.2 to 7.5Enteric Coated (EC) Proton pump inhibitor Granules/Pellets and pH 5.5 to6.2/7.2 to 7.5 Enteric Coated (EC) Symbiotic Granules/Pellets s areblended in desired portions in V-type or similar blender withexcipients. The blended powders are compressed into soft tablets usingtableting equipment.

Example

Final Product—Tablets/Microtablets—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Microcrystallinecellulose - filler, 53% polyvinylpyrrolidone - binder, pregelatinizedstarch - disintegrant and silicon dioxide - flow aid, magnesiumstearate - lubricant) HPMC or equivalent “polymers” (Film coat) 2%Water/Solvents as required 0%

The pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets and pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions in V-type orsimilar blender with excipients to aid in flow, disintegration andlubrication (for tableting machine). The blended powders are compressedinto Tablets/Microtablets using tableting equipment. The tablets arefurther film coated using aqueous or solvent coating solution in acoating pan or fluid bed dryer using HPMC or equivalent “polymers” (Filmcoat).

Final Product—Tablet (2)—Formulation/Manufacturing Process (at localCMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) Uncoated Proton pump inhibitor Granules/Pellets30% pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/ 15% PelletsExcipients (Microcrystalline cellulose - filler, 43%polyvinylpyrrolidone - binder, pregelatinized starch - disintegrant andsilicon dioxide - flow aid, magnesium stearate - lubricant) “Polymers”(pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent “polymers”(Film coat) 2% Water/Solvents as needed 0%

The above Uncoated Proton pump inhibitor Granules/Pellets and pH 5.5 to6.2 Enteric Coated (EC) Symbiotic Granules/Pellets is blended in desiredportions in V-type or similar blender with excipients to aid in flow,disintegration and lubrication (for tableting machine). The blendedpowders are compressed into tablets using tableting equipment. Thecompressed tablets are coated with pH 7.2 to 7.5 sensitive coating usingaqueous or solvent coating solution of “Polymers” in a coating pan orfluid bed drier/coater with optimized conditions (“EC tablets”). Thetablets are further film coated using aqueous or solvent coatingsolution in a coating pan or fluid bed dryer using HPMC or equivalent“polymers” (Film coat).

Example

Final Product—Tablet-in-Tablet (1)—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Microcrystallinecellulose - filler, 53% polyvinylpyrrolidone - binder, pregelatinizedstarch - disintegrant and silicon dioxide - flow aid, magnesiumstearate - lubricant) HPMC or equivalent “polymers” (Film coat) 2%Water/Solvents as required 0%

The pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets is blended in desired portions in V-type or similarblender with excipients to aid in flow, disintegration and lubrication(for tableting machine). The blended powders are compressed into smalltablets/Microtablets using tableting equipment. The above pH 7.2 to 7.5Enteric Coated (EC) Proton pump inhibitor Granules/Pellets are blendedin desired portions in V-type or similar blender with excipients to aidin flow, disintegration and lubrication (for tableting machine). Theblended powder is compress coated over the small tablets/Microtabletsusing compress coat tableting machine. The tablets are further filmcoated using aqueous or solvent coating solution in a coating pan orfluid bed dryer using HPMC or equivalent “polymers” (Film coat).

Final Product—Tablet-in-Tablet (2)—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) Uncoated Proton pump inhibitor Granules/Pellets25% pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/ 15% PelletsExcipients (Microcrystalline cellulose - filler, 48%polyvinylpyrrolidone - binder, pregelatinized starch - disintegrant andsilicon dioxide - flow aid, magnesium stearate - lubricant) “Polymers”(pH 7.2 to 7.5 sensitive coating) 10% HPMC or equivalent “polymers”(Film coat) 2% Water/Solvents as needed 0%

The above Uncoated Proton pump inhibitor Granules/Pellets is blended indesired portions in V-type or similar blender with additional excipientsto aid in flow, disintegration and lubrication (for tableting machine).The blended powders are compressed into small tablets/Microtablets usingtableting equipment. The pH 5.5 to 6.2 Enteric Coated (EC) SymbioticGranules/Pellets is blended in desired portions in V-type or similarblender with excipients to aid in flow, disintegration and lubrication(for tableting machine). The blended EC Symbiotic Granules/Pellets arecompress coated over the small EC tablets/Microtablets using compresscoat tableting machine. The compressed tablets are coated with pH 7.2 to7.5 sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater with optimizedconditions (“EC tablets”). The tablets are further film coated usingaqueous or solvent coating solution in a coating pan or fluid bed dryerusing HPMC or equivalent “polymers” (Film coat).

Example

Final Product—Tablet-in-Capsule (Hard gelatin)(1)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Microcrystallinecellulose - filler, 45% polyvinylpyrrolidone - binder, pregelatinizedstarch - disintegrant and silicon dioxide - flow aid, magnesiumstearate - lubricant) Hard Gelatin/HPMC Capsules 10%

The pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets and pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions in V-type orsimilar blender with excipients to aid in flow, disintegration andlubrication (for tableting machine). The blended powders are compressedinto Tablets/Microtablets using tableting equipment. The excipients andthe compressed tablets filled into hard gelatin capsules usingspecialized encapsulating equipment.

Example

Final Product—Tablet-in-Capsule (Hard gelatin)(2)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated(EC) Symbiotic 15% Granules/Pellets Excipients (Microcrystallinecellulose - filler, 45% polyvinylpyrrolidone - binder, pregelatinizedstarch - disintegrant and silicon dioxide - flow aid, magnesiumstearate - lubricant) Hard Gelatin/HPMC Capsules 10%

The pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC) SymbioticGranules/Pellets are blended in desired portions in V-type or similarblender with excipients to aid in flow, disintegration and lubrication(for tableting machine). The blended powders are compressed into smalltablets/Microtablets using tableting equipment. The above pH 7.2 to 7.5Enteric Coated (EC) Proton pump inhibitor Granules/Pellets are blendedin desired portions in V-type or similar blender with additionalexcipients to aid in flow, disintegration and lubrication (for tabletingmachine). The blended powder and compressed tablets are filled intolarge Hard Gelatin Capsules using encapsulating equipment.

Example

Final Product—Tablet-in-Capsule (Hard gelatin)(3)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets pH 5.5 to 6.2 Enteric Coated (EC)Symbiotic Granules/ 15% Pellets Excipients (Microcrystalline cellulose -filler, 35% polyvinylpyrrolidone - binder, pregelatinized starch -disintegrant and silicon dioxide - flow aid, magnesium stearate -lubricant) “Polymers” (pH 7.2 to 7.5 sensitive coating) 10% HardGelatin/HPMC Capsules 10% Water/Solvents as required 0%

The pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/Pellets areblended in desired portions in V-type or similar blender with excipientsto aid in flow, disintegration and lubrication (for tableting machine).The blended powders are compressed into small tablets/Microtablets usingtableting equipment. The compressed tablets are coated with pH 7.2 to7.5 sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater with optimizedconditions (“EC tablets”). The above pH 7.2 to 7.5 Enteric Coated (EC)Proton pump inhibitor Granules/Pellets are blended in desired portionsin V-type or similar blender with excipients to aid in flow,disintegration and lubrication (for tableting machine). The blendedpowder and the EC tablets are filled into a larger capsule usingencapsulating equipment.

Example

Final Product—Tablet-in-Capsule (Hard gelatin)(4)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Amount Ingredients (%) Uncoated Proton pump inhibitor Granules/Pellets25% pH 5.5 to 6.2/7.2 To 7.5 Enteric Coated (EC) Symbiotic 15%Granules/Pellets Excipients (Microcrystalline cellulose - filler, 40%polyvinylpyrrolidone - binder, pregelatinized starch - disintegrant andsilicon dioxide - flow aid, magnesium stearate - lubricant) “Polymers”(pH 7.2 to 7.5 sensitive coating) 10% Hard Gelatin/HPMC Capsules 10%Water/Solvents as required 0%

The above Uncoated Proton pump inhibitor Granules/Pellets formulation isblended in desired portions in V-type or similar blender with excipientsto aid in flow, disintegration and lubrication (for tableting machine).The blended powders are compressed into small tablets/Microtablets usingtableting equipment. The compressed tablets are coated with pH 7.2 to7.5 sensitive coating using aqueous or solvent coating solution of“Polymers” in a coating pan or fluid bed drier/coater with optimizedconditions (“EC tablets”). The pH 5.5 to 6.2 Enteric Coated (EC)Symbiotic Granules/Pellets are blended in desired portions in V-type orsimilar blender with excipients to aid in flow, disintegration andlubrication (for tableting machine). The blended powder and the small ECtablets are filled into a larger capsule using encapsulating equipment.

Final Product—Tablet-in-Capsule Hard Gelatin(5)—Formulation/Manufacturing Process (at local CMO, controlled roomtemperature, humidity and oxygen conditions throughout the process):

Ingredients Amount (%) Uncoated Proton pump inhibitor Granules/Pellets25% pH 5.5 to 6.2 Enteric Coated (EC) Symbiotic Granules/ 15% PelletsExcipients (Microcrystalline cellulose - filler, 38%polyvinylpyrrolidone - binder, pregelatinized starch - disintegrant andsilicon dioxide - flow aid, magnesium stearate - lubricant) “Polymers”(pH 7.2 to 7.5 sensitive coating) 10% Hard Gelatin/HPMC Capsules 10%HPMC or equivalent “polymers” (Film coat) 2% Water/Solvents as needed 0%

The above Uncoated Proton pump inhibitor Granules/Pellets is blended indesired portions in V-type or similar blender with additional excipientsto aid in flow, disintegration and lubrication (for tableting machine).The blended powders are compressed into small tablets/Microtablets usingtableting equipment. The pH 5.5 to 6.2 Enteric Coated (EC) SymbioticGranules/Pellets is blended in desired portions in V-type or similarblender with excipients to aid in flow, disintegration and lubrication(for tableting machine). The blended EC Symbiotic Granules/Pellets alongwith the proton pump inhibitor small compressed tablets are filled intolarger capsules using encapsulating machine. The large capsules arecoated with pH 7.2 to 7.5 sensitive coating using aqueous or solventcoating solution of “Polymers” in a coating pan or fluid beddrier/coater with optimized conditions (“EC tablets”). The capsules arefurther film coated using aqueous or solvent coating solution in acoating pan or fluid bed dryer using HPMC or equivalent “polymers” (Filmcoat).

Example

Final Product—Bi-Layer Tablets—Formulation/Manufacturing Process (atlocal CMO, controlled room temperature, humidity and oxygen conditionsthroughout the process):

Ingredients Amount (%) pH 7.2 to 7.5 Enteric Coated (EC) Proton pumpinhibitor 30% Granules/Pellets Excipients (Microcrystalline cellulose -filler, 53% polyvinylpyrrolidone - binder, pregelatinized starch -disintegrant and silicon dioxide - flow aid, magnesium stearate -lubricant) pH 5.5 to 6.2/7.2 to 7.5 Enteric Coated (EC) Symbiotic 15%Granules/Pellets HPMC or equivalent “polymers” (Film coat) 2%Water/Solvents as required 0%

The above pH 7.2 to 7.5 Enteric Coated (EC) Proton pump inhibitorGranules/Pellets are blended in desired portions in V-type or similarblender with excipients to aid in flow, disintegration and lubrication(for tableting machine). The blended powders are compressed into tabletsusing bi-layer tableting equipment (“EC Tablets”). The pH 5.5 to 6.2/7.2to 7.5 Enteric Coated (EC) Symbiotic Granules/Pellets are blended indesired portions in V-type or similar blender with excipients to aid inflow, disintegration and lubrication (for tableting machine). Theblended powder is compressed over the EC tablets using bilayer tabletingmachine. The tablets are further film coated using aqueous or solventcoating solution in a coating pan or fluid bed dryer using HPMC orequivalent “polymers” (Film coat).

Final Product Packaging (at local CMO, dry low humidity and low oxygen(N2 purging) conditions throughout the process):

The above granules are packaged in sachet, and the coated tablets,capsules are packaged into bottles with induction sealing or blisteredat low humidity (at or below 40% RH) and controlled room temperatureconditions (at 20 to 25 degrees C.).

Quality Control Release Testing (Active Pharmaceutical Ingredient (API)and Final Drug Product) Symbiotic.

Test Methods and Assessment Description Granules, pellets, tablets,capsules in blisters or bottles or sachets Appearance Visual inspectionfor color, shape, etc. Identification Genes, species, strains.Morphological appearance via Microscopic evaluation and /or multiplexPCR as well as other tests including biochemical methods such asfermentation profile or genotypic methods, e.g. ribotyping, restrictionfragment length polymorphism (RFLP), or both. In addition, develop aspecific identity assay for critical biological activity. Others testmay include: DNA-DNA hybridization to specify strains in species; DNAsequence coding per WHO; Strain typing include Pulsed Field Gelelectrophoresis (PFGE), etc. Potency - Viable Microscopic testing, orOpacity to measure viable cells per organisms unit or dose, i.e. colonyforming units (CFU) Potency Assay Assessment of CFU (on solid medium)and tests to correlating with activity. M-viability plating. PurityEndotoxin content, antibiotic residue and/or the quantification ofresidual toxic components or contaminants introduced during manufactureby Elisa or amino acid profile Microbial bioburden or Extraneousmaterials including pathogens by using Elisa or contaminants and limitsamino acid profile or SDS page or ion exchange (related substances)chromatography, etc. Microbial limits by US Pharmacopeia (USP 31 <61>).Percent viable cells Micro testing after regrown in appropriate mediaand test, e.g., Dead/live assay by ATP. Also determination of non-viableunits per g i.e., by electro-zone count of non-fluorescent cells (SDSPAGE) Particulate matter USP 31 <788> Pyrogens TBD pH Testing pH meterResidual moisture Water content, USP 31 <921> Content Uniformity ATPLive/Dead Assay ATP Heavy metals Inductively Coupled Plasma-AtomicEmission Spectrophotometry (ICP-AES); Inductively Coupled Plasma- MassSpectroscopy (ICP-MS); Atomic Emission Spectrophotometry (AES); orAtomic Absorption Spectrophotometry (AAS). Water content Karl FischerPackage Integrity Leaker test by vacuum Stability Potency, viable celldetermination, microbial contamination, pH an residual moisture

Proton Pump Inhibitor(s)

Test Methods and Assessment Identification HPLC and other Assay HPLC andother Impurities and Related sub HPLC and other Content uniformity HPLCand other Microbial limits US Pharmacopeia (USP 31 <61>).

Symbiotic and Proton Pump Inhibitor

Test Methods and Assessment In-vitro release testing (via USP paddle orbasket dissolution testing Medium: pH 1 buffer (simulated gastric), pH 6buffer, pH equipment): 7.2 to 7.5 buffer (simulated intestinal fluid),followed by pH 5.5-6.2 buffer (simulated colonic fluid). Sample Times:pH 1 buffer - 1 hour pH 6 buffer - 1 hour pH 7.2 to 7.5 - 1, 2, 3 and 4hours pH 5.5 to 6.2 - 1, 2, 4 and 8 hours Symbiotic Assay: Microbiologytesting for count (cfu/gram) for Proton pump inhibitor Assay: HPLCStability testing (0, 6, 12, Symbiotic: 18 and 24 months):Identification, Appearance, Potency, viable cell determination,microbial contamination, pH and residual moisture, related substance,water content, Live/dead Assay, etc. Proton pump inhibitor:Identification, Assay, Impurities, Related Substances, microbialcontamination, pH and residual moisture, IVRT, etc.

Example 4

Oral delivery of biologic and non-biologic drugs to distal ileum and/orcolon.

The pill-in-pill dosage form (e.g., tablet-in-tablet orcapsule-in-capsule, etc.) would pass through the GI tract from stomach(pH 1 to 4), to duodenum (pH 5.5 to 6.2) and deliver to distal ileum (pH7.3 to 8.0) and/or proximal colon (pH 5.5 to 6.2), depending on thedesign. The release from this pill-in-pill dosage form would not requireany other aid (e.g. sugars, starches etc.) or external conditions orenergy source such as presence or absence of enzymes or bacterial florain the distal ileum or proximal colon. Another advantage of thepill-in-pill dosage form would be that the drug release is possible invarious disease conditions (e.g. IBD, etc.) when the pH of the distalileum and proximal colon may have significantly different from thenormal values of above pH 7.4 and below pH 6.5, respectively. In orderto demonstrate the concept, the tablet was initially developed andfollowed by the capsule-in-capsule dosage form (See Table 1 below) todeliver a small molecule or biologic directly to the proximal colon,within a 2 hour delivery target window, bypassing the stomach (2 hours)and duodenum (1 hour) and the ileum (2 hours).

TABLE 1 Capsule-in-Capsules Design Inner Capsule Outer Capsule Materialand Gelatin or HPMC or other, Gelatin or HPMC or other, larger Size ofsmaller size, e.g. smaller than size, e.g. larger than #1, etc. Capsule#3, etc. Band sealed for coating Band sealed for coating (with or (withor without seal coat) without seal coat) API (biologic Small molecules,prokaryotes Small molecules, prokaryotes and non- cells (e.g. archaea,bacteria), cells (e.g. archaea, bacteria), biologic drugs) eukaryotecells (e.g. fungus, eukaryote cells (e.g. fungus, plants), virusparticles, proteins,, plants) virus particles, proteins, peptides,parasites, vaccine cells, peptides, parasites, vaccine antigens, etc. ornothing antigens, etc., or nothing Excipients Prebiotics, solids,liquids, semi- Prebiotics, solids, liquids, semi- solids, growthpromoters solids, growth promoters Dosage forms Formulated tablets, softand hard Formulated tablets, soft and hard capsules, pellets, powders,etc. capsules, pellets, powders, etc. Coatings Reverse enteric with orwithout Regular enteric with or without fillers fillers Target releaseAt pH 6.5 (or below) within 2 At pH 7.0 (or above) within 2 hoursdelivery target window hours delivery target window (goal: proximalcolon delivery) (goal: distal ileum delivery) Testing Standard USPdissolution testing in various multiple media

The target release for drugs in dissolution media was pH 6.5 in 2 hours,representing the proximal colon and with no release at pH 1.2 (gastric)for 2 hours, pH 5.5 (duodenal) for 1 hour, pH 7.0 (ileum) for 1 hour andpH 7.4 (distal ileum) for 1 hour. Probiotic and acetaminophen were usedas representative biologic agent (lyophilized bacteria) and smallmolecule, respectively. Acetaminophen was also used as a marker forprobiotic during release testing. The small molecule and the probioticmixes were prepared separately with and without additional excipients.Hydroxypropylmethyl cellulose (HPMC) capsules were used as the reservoirfor carrying these drugs. HPMC capsules have several advantages as theyare made from non-animal materials, chemically stable, have low moisturecontent (protect lyophilized bacteria), less brittle even at lowhumidity (survive the GI transit), fast dissolution, biodegradable, nocrosslinking and suitable to automatic capsules filling machines. Thesecapsules can be band sealed, which has the following advantages: avoidthe need for additional steps of seal coating with polymers; avoid theneed for excess moisture and heat required for processing, especiallyimportant for maintenance of the viability of the biologicals; andminimize the impact on release of drug from the capsules.

The polymers evaluated were aqueous based methacrylic acid copolymersand were designated as either reverse enteric (e.g. EUDRAGIT® E PO) orregular enteric (e.g. EUDRAGIT® FS 30D, EUDRAGIT® S100, EUDRAGIT® L100,EUDRAGIT® L30D-55) alone or in combinations. EUDRAGIT® E PO is designedto solubilize at pH 6.5 or below and also possess good moisture barrierproperties which protected lyophilized bacteria and further improvedstability. EUDRAGIT® FS 30D, EUDRAGIT® S100, EUDRAGIT® L100, EUDRAGIT®L30D-55 are designed to solubilize above pH 7.0, 6.5, 6.0, 5.5,respectively. These polymers can be applied on the tablets and capsuleswith heat and moisture below, 30° C. and 40% RH, respectively, which isimportant for the maintenance of the viability of biological drugs. Thetablet dosage form was initiated and then followed by thecapsule-in-capsule dosage form. These capsules were subject to standardUSP dissolution testing. Notably, these similar principles apply fordelivery to distal ileum alone and in combination with proximal colon.Applications of this technology would broadly include the delivery ofMicrobiome Ecology Therapy (MET); Small molecule drugs and Vaccines,etc.

Initial development focused on coating of the acetaminophen (APAP) coretablets using APAP as the marker for monitoring the release of biologicand non-biologic small molecule drugs from the dosage form. The 325 mguncoated tablet cores dissolved fairly rapidly, greater than 85% in 45minutes in USP dissolution apparatus with basket at 50 rpm in pH 6.5phosphate buffer (Formulation 1, FIG. 8). When these APAP tablets werecoated with reverse enteric material (Evonik EPO) at up to 18 mg/cm²level and performed the dissolution testing under the same conditions,100% of the APAP was released at target pH 6.5 within 2 hours,simulating the release of the drug in the proximal colon (Formulation 2,FIG. 9). Since this coating normally was designed to dissolve below pH6.5, the rate of release from the tablet formulation was more rapid atpH 6.0, as expected. Also as expected, no release of APAP was observedfrom the tablets at pH 6.8, pH 7.0 and pH 7.4.

The 325 mg uncoated tablet cores dissolved fairly rapidly, greater than85% in 45 minutes in USP dissolution apparatus with basket at 50 rpm inpH 7.0 phosphate buffer (Formulation 1, FIG. 10). These APAP tabletswere coated with regular enteric material (Evonik FS30D/L30 Mixtures) atup to 15 mg/cm² level, and subject to dissolution in pH 1.2 for 2 hours,pH 5.5 for 1 hour and pH 7.0 for 2 hours using the same apparatus andspeed. The formulation passed the performance test in pH 1.2 for 2 hoursand 1 hour at pH 5.5. The release rate at pH 7 was slower and did notpass the 2 hour test. However, the release rate increased as expectedwith lower ratio of Evonik FS30D/L30, e.g. 50/50 (Formulations 3 (a-c),FIG. 11). Based on these results, it was concluded that more permeablecoatings would be required to obtain the desired release profiles in pH7.0. Additional optimization would also be required for the tabletdosage form including consideration of other formulation factors, suchas coating thickness, total polymer applied, physico-chemical propertiesof the drug, loading dose, size and shape of the tablets, etc.

As indicated earlier, the aim was to develop a capsule-in-capsule dosageform which would deliver a small molecule or a biologic to the proximalcolon, within a 2 hour delivery target window, without the need foradditional compression and also for ease of demonstrating theapplications of colonic drug delivery technology. The principlesdeveloped here can be easily adapted to other dosage forms, such ascompressed tablets, pellets, oral disintegrating tablets, liquid filledcapsules, etc.

The uncoated inner smaller capsule containing APAP was subjected to USPdissolution tests with basket at 75 rpm and paddle at 50 rpm in pH 6.5dissolution media. The release from the capsules was much slower(Formulation 4, FIG. 12) as compared to the tablets and higher speedswould be required to disintegrate the capsules in the basket. However,there was almost no difference between the release profiles for thecapsules either in the basket or paddle. Based on physical appearance ofthe capsules in the paddle method, the capsules appear to break downmore easily as compared to the basket method, but were still notcompletely disintegrated. The smaller inner APAP capsules were coatedwith reverse enteric coat, EUDRAGIT® EPO at 10 mg/cm². The coatedcapsules were subjected to USP dissolution tests with basket at 75 rpmand paddle at 100 rpm in pH 6.5 dissolution media. The capsules met therelease requirement at pH 6.5 in 2 hours when using the paddle method at100 rpm (Formulation 5, FIG. 13). Physically all the capsules had brokendown and completely disintegrated. Note there was no release from thecapsules in the basket at 75 rpm and also the capsules were physicallyintact (not broken down or disintegrated) in the basket even after 2hours. The coated capsules were also subject to pH 6.8 dissolution mediafor 2 hours at 10 mg/cm² coating level using the paddle at 100 rpm. Asexpected there was no release from the capsules (Formulation 5, FIG.14). Also, physically, the capsules had not disintegrated. The paddlespeed of 100 rpm for dissolution testing was justified since the releasein vivo is generally associated with a significant gut agitation andcompression, something that may not been seen with the in vitrodissolution test. Also, typically for enteric coated capsules,disintegration apparatus (similar to USP dissolution apparatus III) withhigh turbulence are typically used for evaluation of release.

The larger outer seal coated (no enteric) capsule containing APAP wassubjected to USP dissolution tests with paddle at 100 rpm in pH 6.5dissolution media. The release from these capsules was rapid and allcapsules released the drug within 1 hour (Formulation 6, FIG. 15). Alsophysically all capsules had disintegrated. The larger outer APAPcontaining capsules were coated with regular enteric coat, EUDRAGIT®L100 and L100/S100, 50/50 mix at 7.5 mg/cm². These coated capsules weresubjected to USP dissolution tests with paddle at 100 rpm in pH 1.2 (2hours), pH 5.5 (1 hour), pH 7.0 (1 hour) and pH 7.4 (1 hour) dissolutionmedias. The coated capsules containing L100 alone had slight release dueto drug permeation at pH 5.5 in 1 hour, but otherwise acceptable. Thecoated capsules containing L100/S100 50/50 mix did not pass the releasetest at pH 7.0/7.4 in 2 hours (Formulation 7 (a-b) FIG. 16). Hence thelager outer APAP containing capsules were coated with regular entericcoat, EUDRAGIT® L100/S100, 75/25 mix at 5 and 7.5 mg/cm². These coatedcapsules were subjected to USP dissolution tests with paddle at 100 rpmin pH 1.2 (2 hours), pH 5.5 (1 hour), pH 7.0 (1 hour) and pH 7.4 (1hour) dissolution medias. All the capsules passed the dissolution at pH1.2 for 2 hours. However, the coated capsules containing 7.5 mg/cm² didnot pass the release test at pH 7.0/pH 7.4 over 2 hours. The coatedcapsules containing 5 mg/cm² L100/S100 75/25 mix did pass the releasetest at all the pH conditions (Formulation 8 (a-b), FIG. 17), except forslight permeation of drug at pH 5.5. Hence, applying a slightly highercoating thickness would eliminate this problem for drug release targetedto the distal ileum.

Based on the above results, with the goal of release in the proximalcolon, the smaller capsules containing APAP, band sealed and entericcoated with EUDRAGIT® EPO 10 mg/cm² were filled into larger capsules,band sealed and further coated with EUDRAGIT® L100/S100, 75/25 mix, 5mg/cm² on the outside. These capsule-in-capsules were subject toin-vitro USP dissolution testing, paddle at 100 rpm, for APAP release inpH 1.2 media for 2 hours, pH 5.5 for 1 hour, pH 7.0 for 1 hour, pH 7.4for 1 hour, pH 6.5 (phosphate) for 2 hours. The results confirm fullAPAP release specifically at pH 6.5 within 2 hours from the innercapsule, and with no release at pH 1.2 for 2 hours, pH 5.5 for 1 hour,pH 7.0 for 1 hour and pH 7.4 for 1 hour. (Formulation 9, FIG. 18)Physically the outer capsules remained intact with no disintegration atpH 1.2 for 2 hours and pH 5.5 for 1 hour. Then the outer capsulescompletely disintegrated after exposure to pH 7.0 for 1 hour and pH 7.4for 1 hour, and the inner capsule was observed and it had physicallyremained intact. The inner capsules then completely disintegrated whenexposed to the pH 6.5 media within 2 hours. The physical observationsare very consistent with the drug release data reported in FIG. 18.

Similar to the APAP capsule-in-capsules, the smaller probioticcontaining capsules, were band sealed and enteric coated with EUDRAGIT®EPO 10 mg/cm² and were filled into larger capsules, band sealed andfurther coated with EUDRAGIT® L100/S100, 75/25 mix at 5 mg/cm² on theoutside. These capsule-in-capsules were subject to USP dissolutiontesting, paddle at 100 rpm, for probiotic bacteria release in pH 1.2media for 2 hours, pH 5.5 for 1 hour, pH 7.0 for 1 hour, pH 7.4 for 1hour, pH 6.5 for 2 hours (saline phosphate buffer). Saline buffer wasused to maintain isotonicity of the dissolution medium and ensureviability of the lyophilized bacteria once they are exposed to theaqueous solution. Physically, these probiotic capsules behaved exactlyin the same manner as the APAP capsules. It could be surmised inferredthe bacteria would be released from probiotic capsules exactly in thesame manner as the APAP from the APAP capsules, i.e. full release at pH6.5 within 2 hours from the inner capsule, and no release at pH 1.2 for2 hours, pH 5.5 for 1 hour, pH 7.0 for 1 hour and pH 7.4 for 1 hour.

-   -   Based on SEM evaluations of reverse and regular coatings, the a        preferable coating level thickness is:    -   First capsule (inner pill)—EUDRAGIT® EPO, 5 mg/cm²-10 mg/cm²:    -   60-180 microns (μm) for size #3 capsule    -   Second capsule (outer pill)—EUDRAGIT® L100/S100 (75/25)—5        mg/cm²-10 mg/cm²    -   60-180 microns (μm) for size #0 capsule

The uncoated and coated CIC capsules were analyzed to determine thelevel of degradation due to processing. The data suggested, and shown inTable 2, that the total strain count as measured by total CFU percapsules did not change significantly. Hence the process of handling,banding and coating applications, storage and shipment did not have anysignificant effect on viability of the 3 bacteria strains tested in theformulations, including the aerobic strains of S. thermophilus and L.acidophilus and anaerobic strains of B. longum

Materials and Methods

Acetaminophen (APAP):

-   -   (Receiving # RCA31252; Guardian Drugs) Malinckrondt Inc.—lot        #784513B054-3% PVP granulated powder for tableting.    -   Aacetaminophen (Paracetamol, USP-APC-150)—ALP Co. (China)—Lot        #0908302. Acetaminophen (APAP) 325 mg core tablets (Lot #        L0577-215-Guardian Drugs, NJ)

Probiotic Capsule: Azodyl (size #3) (Batch #023042-20; Lot #5241113;Kibow Biotech; Newtown Square, Pa. 19073)

HPMC Capsules:

-   -   Qualicaps Size #3/S-LOK—Lot # E1305982-Clear VAA (cap & body)    -   Qualicaps Size #3/S-LOK—Lot # E1205667-Op. White XAK (cap &        body)    -   Qualicaps Size #3/S-LOK—Lot # E1106719-Op. Brown 15 XJX (cap &        body)    -   Qualicaps Size #0/S-LOK—Lot # E1101410-Op. White XAK (cap &        body)    -   Qualicaps Size #0/S-LOK—Lot # E1106476-Op. Brown 15 XJX (cap &        body)

Methacrylic Acid Co-Polymers for Coating:

-   -   EPO-Ready Mix-Evonik-lot# H131181012    -   EUDRAGIT®-L30D 55-Evonik—Lot# B130514207    -   EUDRAGIT®-FS 30D Evonik—Lot# B130365004    -   EUDRAGIT®-S100-Evonik—Lot# B100405198    -   EUDRAGIT®-L100-Evonik—Lot# B120603009    -   Plasacryl T20-Evonik—Lot # PT130705

Coating Polymers:

-   -   HPMC ES-Dow—Lot # YG040124L1

Plasticizers:

-   -   Triethyl Citrate (TEC)-Vertellus—Lot 3 132530

Surfactants:

-   -   Polyethylene Glycol 4000-AlfaAesar—Lot #10167045    -   Polysorbate 80 (Tween 80)-BASF—Lot#3158092

Other Excipients:

-   -   Talc-Brenntag—Lot #410052-43    -   Microcrystalling cellulose (MCC)-MC-102-Blanver—Lot3 135002006    -   Lactose Monohydrate (Supertab 11SD)-DFE Pharma—Lot#10677724    -   Pre-gelatinized Starch-DFE Pharma—Lot #-10601223    -   Crospovidone-QJNI Co.,—Btch #20130115    -   DiCalcium Phosphate-Innophos—Lot#0701047    -   Coloidal silicon dioxide (Aerosil-200)-Evonik—Btch #1012082200    -   Silicon dioxide (Aerosil R972)-Degussa—Lot#3158092923    -   Magnesium Stearate-FACI Asia-Batch # MGSP0216    -   Magnesium Stearate-Mallinkrodt—Lot#-071226.    -   Hydroxy-propyl Methyl Cellulose-Shinogi—Lot #90936C

Chemicals:

-   -   Ammonium Hydroxide-AlfaAesar—Lot # E302012    -   Ethyl Alcohol-Fischer—Lot # M02539    -   Potassium Dihydroigen Phosphate-Alfa Aesar—Lot #1013774    -   Sodium Hydroxide-Macron Chemicals—Batch 98#0000039706

Method for Prepare Core Tablets and Compression

The required amount as shown in the formula A of APAP, MCC,Pre-Gelatinized Starch, Crospovidone & Colloidal Silicon di-oxide waspassed thru #20 sieve and was loaded in a suitable belnder and mixed for25 minutes. At the end of the process the Magnesium stearate was addedand blend was mixed for additional 5 minutes. At the end of the processthe material was unloaded into clean poly-lined containers. The blend(100 kg) was compressed on a Korsch XL-100 10 station press. Amodified-oval shaped, standard concave tooling (16.5 mm×7.5 mm) havingplain surfaces (no logo) on both sides was used. This was design waschosen based on providing suitable substrate for functional coating.Tablets were compressed to a target weight of 600 mg (containing 325 mgAPAP) with Friability of NMT 1% and Hardness of >24 kP. The tabletweight, thickness, hardness and friability was monitored as in-processtest throughout the batch manufacturing. Tablet samples were taken toensure disintegration time was <5 min.

Encapsulation and Banding

Encapsulation of Formulation B:

All the ingredients were passed thru a MMC Co-mill to ensure noagglomerates were present in the blend. A 8 Qt V-Blender was used to mixall the ingredients except Magnesium stearate. After mixing all theingredients Magnesium stearate was added. The blend was mixed for 2additional minutes before discharge into a double-poly lined container.The index K120i (S/N 0963-27) was set-up to run the capsules (size #3)from Qualicaps. The capsule polisher (Model TG-20) and weight scales(Mettler Toledo Scale) were set-up appropriately for the run. Theprocessing room temperature and humidity log was documented for the run.In-process weight samples were collected during the run to ensure thetarget weight is achieved. The capsules were polished and collected in adouble-lined poly-bags in container.

Encapsulation of Formulation D:

A FastLock K200F with vibration table was used for filling the size #0capsules from formulation D. Size #0 Quali V capsules were used to fillthe 3% granulated APAP powder. Approximate 100 capsules were filled eachtime. The weights for capsules were recorded.

Banding of Capsules:

Banding of capsules was performed on the IMA Bander (BD 1723) Typicallybanding of capsules results in a weight gain of 1-1.5 mg which is withinthe weight variation of the capsules so it is typically considered apart of the capsule weights and the associated variations.

Preparation of Spraying Dispersions

Preparation of EUDRAGIT®-EPO Ready Mix:

The Ready mix is a standard coating system from Evonik which has 51% EPOpolymer. About 150 g of this dry mix is added to about 850 g of water togive approximately 1 kg of spray suspension. The material is mixed usinga high shear mixer for approx. 30 minutes. The entire suspension is thenpassed through a 0.5 mm sieve. The suspension is next ready for sprayingto the substrate using typical standard processing parameters.

Preparation of L-30D 55:

For 1 kg of spray suspension approx. 570 g of EUDRAGIT® L30D 55dispersion is added in a larger mixer vessel. Approx. 145.5 g ofPlasacryl HTP20 (anti-tacking/plasticizer system) is added to the mix.The suspension is diluted with required amount of water to get 1 kg ofspray dispersion. The PlasAcryl need to be shaken before transfer to anyvessel. The entire suspension is stirred for 10 minutes using apropeller stirrer. The entire suspension is passed through a 0.5 mmsieve. The suspension is next ready for spraying to the substrate usingtypical standard processing parameters.

Preparation of FS 30 D:

For 1 kg of spray suspension approx. 606.1 g of EUDRAGIT® FS30Ddispersion is added in a larger mixer vessel. Approx. 90.9 g ofPlasacryl HTP20 (anti-tacking/plasticizer system) is added to the mix.The suspension is diluted with required amount of water to get 1 kg ofspray dispersion. The PlasAcryl need to be shaken before transfer to anyvessel. The entire suspension is stirred for 10 minutes using apropeller stirrer. The entire suspension is passed through a 0.5 mmsieve. The suspension is next ready for spraying to the substrate usingtypical standard processing parameters.

Preparation of L100 Dispersion:

For 1 kg of spray suspension approx. 99.5 g of EUDRAGIT® L100 was addedinto ⅔ rd of the water and stir for approximately 5 minutes and makingsure the powder is all wetted. Add 1N NH₃ (56 g) slowly into theEUDRAGIT® suspension and stir for approximatly 60 minutes. Add 49.8 g ofTriethyl citrate (TEC) and stir for additional 60 minutes. Separately,homogenize 49.8 g of Talc with the remaining amount (⅓ rd) of water for10 minutes using a high shear mixer. Pour the talc suspension into theEUDRAGIT® dispersion while stirring with a conventional stirrer. Theentire suspension is passed through a 0.5 mm sieve. The suspension isnext ready for spraying to the substrate using typical standardprocessing parameters.

Preparation of S100 Dispersion:

For 1 kg of spray suspension approximately 99.4 g of EUDRAGIT® S100 isadded into ⅔ rd of the water and stirred for approximately 5 minutes andmaking sure the powder is all wetted. Add 1N NH₃ (67.5 g) slowly intothe EUDRAGIT® suspension and stir for approximately 60 minutes. Add 49.7g of Triethyl citrate (TEC) and stir for additional 60 minutes.Separately, homogenize 49.7 g of Talc with the remaining amount (⅓ rd)of water for 10 minutes using a high shear mixer. Pour the talcsuspension into the EUDRAGIT® dispersion while stirring with aconventional stirrer. The entire suspension is passed through a 0.5 mmsieve. The suspension is next ready for spraying to the substrate usingtypical standard processing parameters.

For mixtures of two components prepare them separately and then add asper the desired ratios.

Coating of the Tablets and Capsules

All coatings of tablets and capsules were performed on the ThomasEngineering Accela Cota Compu-Lab-24-190. The formulations were coatedin a 12″ pan with two baffles. A minimum batch size of 400 g was usedfor the coatings. For some formulations a larger batch size of 700-1500g was processed. A single Schlick gun (970/7-1 75S) with a nozzle sizefrom 0.8-1.2 mm depending on the batch size and the flow rate of thesuspension was used. The processing conditions were varied depending onthe batch size and the coating material used. For each type of coatingspecific processing conditions were followed. For the safety of theproduct, the product temperature was always maintained below 30° C.

The general processing parameters used broadly is as follows:

Inlet air temp: 30-40° C.

Exhaust Temperature—25-30° C.

Product Temperature: 24-29° C.

Inlet air flow:100-300 CFM

Pump speeds: 2.5-20 rpm

Atomization air pressure:10-30 psi

ID of the tubing used: 3.2 mm,

Pan speed: 4-15 rpm.

Dissolutions testing:

Disintegration apparatus, dissolution apparatus, baskets, paddles andspeeds, temperature and dissolution media, Assay, HPLC, CFU forprobiotics, sampling scheme. The inner capsules were subject todissolution testing at pH 6.5 or pH 6.8 phosphate buffers for up to 2hours. The outer capsules were subject to dissolution media at pH 1.2for 2 hours, pH 5.5 for 1 hour, pH 7.0 for 1 hour and pH 7.4 for 1 hour.The combined capsules were subject to dissolution media at pH 1.2 for 2hours, pH 5.5 for 1 hour, pH 7.0 for 1 hour, pH 7.4 for 1 hour, pH 6.5for 2 hours (with saline as isotonic agent for probiotic.

Analysis of Probiotic Capsules Before and after Coating

The contents of capsules were aseptically transferred into a sterilebottle. The two capsule contents were dissolved in saline. A sample wasdrawn for enumeration and incubated at 37° C. After 3 days of incubationat 37° C. (aerobically for S. thermophilus and L. acidophilus,anaerobically for B. longum) the colonies were counted in triplicate.

TABLE 2 Strain count (CFUs in billions), pre and post coating ofcapsules Uncoated capsules Coated capsules Average (range) Strain CountStrains (CFU in billions), n = 3 S. thermophiles*  13.5 (12.5-15.5) 14.2(13.5-14.5) L. acidophilus* 2.6 (2.2-3.3) 1.8 (1.25-2.1) B. longum** 2.6(2.0-3.1)  1.9 (1.65-2.15) Total Count 18.7 17.9 *Aerobic **Anaerbic

Formulations:

Formulation 1: 325 mg APAP Tablets Core

Amount Ingredients mg % Acetaminophen (APAP)-3% PVP 335 56 granulatedform for tableting Microcrystalline Cellulose, USP 225 37Pre-gelatinized Starch 18 3 Crosspovidone 18 3 Colloidal silicon dioxide3 <1 Magnesium stearate 1 <1 Total 600 100

Formulation 2: APAP Tablets 325 mg—Sealed with 4 mg/cm² Seal (HPMC)Coated with EUDRAGIT® EPO 18 cm²

Amount Ingredients mg % Core Tablet: Acetaminophen 325 mg tablet 606 78(Formulation 1) above 4% w/w Seal Coating: HPMC E5 20 3 PEG 6000, USP 3<1 Water, qs (removed from formulation) — — Functional coating (18mg/cm²): EUDRAGIT ® EPO Readymix 147 19 Water, qs (removed fromformulation) — — Total 776 100

Formulation 3a: APAP Tablets 325 mg, Seal Coated with 4% HPMC & EntericCoated with FS30:L30D55 (90:10), 7.5 mg/cm²

Amount Ingredients mg % Core Tablet: Acetaminophen 325 mg tablet 606 89(Formulation 1) above Seal Coating (4%): HPMC E5 20 3 PEG 6000, USP 4 <1Water, qs (removed from formulation) — Functional coating (90:10) 7.5mg/cm² EUDRAGIT ® FS30D 44 6 EUDRAGIT ® L30D55 4 <1 Plasacryl 5 <1Water, qs (removed from formulation) — Total 684 100

Formulation 3b: APAP Tablets 325 mg, Seal Coated with 4% HPMC & EntericCoated with FS30:L30D55 (75:25), 7.5 mg/cm²

Amount Ingredients mg % Core Tablet: Acetaminophen 325 mg tablet 606 89(Formulation 1) above Seal Coating (4%): HPMC E5 20 3 PEG 6000, USP 4 <1Water, qs (removed from formulation) — Functional coating (75:25) 7.5mg/cm² EUDRAGIT ® FS30D 37 5 EUDRAGIT ® L30D55 12 2 Plasacryl 5 <1 TEC 1<1 Water, qs (removed from formulation) — Total 685 100

Formulation 3c: APAP Tablets 325 mg, Seal Coated with 4% HPMC & EntericCoated with FS30:L30D55 (50:50), 7.5 mg/cm²

Amount Ingredients mg % Core Tablet: Acetaminophen 325 mg tablet 606 87(Formulation 1) above Seal Coating (4%): HPMC E5 19 3 PEG 6000, USP 4 <1Water, qs (removed from formulation) — Functional coating (50:50) 7.5mg/cm² EUDRAGIT ® FS30D 25 4 EUDRAGIT ® L30D55 25 4 Plasacryl 5 1 TEC 1<1 Water, qs (removed from formulation) — Total 685 100

Formulation 4: Composition of Uncoated APAP Capsules (Size #3)

Amount Ingredients mg % Acetaminophen (APAP) powder* 91 52 Lactose, USP72 41 DiCalcium Phosphate 2 1 Colloidla silicon dioxide 5 3 Magnesiumstearate 5 3 Av. Wt. of Size # 3 HPMC Quali V capsules 51 Total 226 100

Formulation 5: Composition APAP Capsules Coated with EUDRAGIT® EPO, 10mg/cm²

Amount Ingredients mg % Uncoated 91 mg APAP capsule 226 86 (Formulation4) Functional coating (10 mg/cm²): EUDRAGIT ® EPO Readymix 37 14 Water,qs (removed from formulation) — Total 263 100

Formulation 6: APAP Capsules (Size #0) Seal Coated with HPMC, 6 mg/cm²

Amount Ingredients mg % Acetaminophen (3% PVP granulation) APAP 346 72powder Av. Wt. of Size # 0 HPMC capsules 105 22 Total wt. of uncoatedsize # 0 APAP capsules 451 Seal Coating (6 mg/cm²): HPMC E5 25 5 PEG6000 4 <1 Water q.s. (removed from the formulation) — Total 480 100

Formulation 7 (a): APAP Capsules (Size #0) Seal Coated with HPMC, 6mg/cm² and Enteric Coated with EUDRAGIT® L100, 7.5 mg/cm²

Ingredients mg Amount (%) APAP capsule (size #0), seal coated(Formulation 6) 480 86 Functional coating (7.5 mg/cm²): EUDRAGIT ® L10039 8 TEC 19 3 Talc 19 3 Total 557 100%

Formulation 7 (b): APAP Capsules (Size #0) Seal Coated with HPMC, 6mg/cm² and Enteric Coated with EUDRAGIT® L100/EUDRAGIT® S100 (50/50) 7.5mg/cm²

Ingredients mg Amount (%) APAP capsule (size #0), seal coated(Formulation 6) 480 87 Functional coating (7.5 mg/cm²): EUDRAGIT ® S10018 4 EUDRAGIT ® L100 18 4 TEC 18 3 Talc 17 2 Total 551 100

Formulation 8 (a). APAP Capsules (Size #0) Seal Coated with HPMC, 6mg/cm² and Enteric Coated with EUDRAGIT® L100/EUDRAGIT® S100 (75/25) 5mg/cm²

Ingredients mg Amount (%) APAP capsule (size #0), seal coated(Formulation 6) 480 87 Functional coating (5 mg/cm²) (75:25) EUDRAGIT ®L100 18 4 EUDRAGIT ® S100 6 1 TEC 13 4 Talc 12 4 Total 529 100

Formulation 8 (b). APAP capsules (size #0) seal coated with HPMC, 6mg/cm² and enteric coated with EUDRAGIT® L100/EUDRAGIT® S100 (75/25) 7.5mg/cm²

Ingredients mg Amount (%) APAP capsule (size #0), seal coated(Formulation 6) 480 87 Functional coating (7.5 mg/cm²) (75:25)EUDRAGIT ® L100 27 5 EUDRAGIT ® S100 9 2 TEC 17 3 Talc 18 3 Total 551100

Formulation 9: APAP Capsule-in-Capsule (CIC) [Inner Capsule (Size#3)Enteric Coated with EUDRAGIT® EPO, 10 mg/cm²; and Outer Capsule (Size#0) Enteric Coated EUDRAGIT® L100/S100 75/25, 5 mg/cm²

Amount Ingredients mg (%) Acetaminophen (3% PVP granulation) APAP powder155 38 Av. Wt. of Size # 3 HPMC capsules 51 13 Total wt. of bandeduncoated size # 3 APAP capsules 206 Inner coating (10 mg/cm²):EUDRAGIT ® EPO ready mix 53 13 Water qs. (removed from formulation) —Total wt. of coated size # 3 APAP capsule 259 Wt. of size # 0 capsule &banding 99 25 Wt. of banded uncoated CIC (size # 0) filled with 358 size# 3 coated APAP (~151 mg) capsule Outer coating (5 mg/cm²): EUDRAGIT ®L100 18 4 EUDRAGIT ® S100 6 1 TEC 12 3 Talc 12 3 Total 406 100

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Accordingly, the presentinvention is not limited except as by the appended claims.

All patents, patent applications, publications, scientific articles, websites, and other documents and materials referenced or mentioned hereinare indicative of the levels of skill of those skilled in the art towhich the invention pertains, and each such referenced document andmaterial is hereby incorporated by reference to the same extent as if ithad been incorporated by reference in its entirety individually or setforth herein in its entirety. Additionally, all claims in thisapplication, and all priority applications, including but not limited tooriginal claims, are hereby incorporated in their entirety into, andform a part of, the written description of the invention. Applicantsreserve the right to physically incorporate into this specification anyand all materials and information from any such patents, applications,publications, scientific articles, web sites, electronically availableinformation, and other referenced materials or documents. Applicantsreserve the right to physically incorporate into any part of thisdocument, including any part of the written description, the claimsreferred to above including but not limited to any original claims.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of” may be replaced with either of the other two terms inthe specification. Also, the terms “comprising”, “including”,containing”, etc. are to be read expansively and without limitation. Themethods and processes illustratively described herein suitably may bepracticed in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. It is also that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality (for example, a culture or population)of such host cells, and so forth. Under no circumstances may the patentbe interpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresreported and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

REFERENCES

The contents of all references cited herein are incorporated byreference herein for all purposes.

-   1. Hajishengallis G, Darveau R P, Curtis M A. The keystone-pathogen    hypothesis. Nat Rev Microbiol. 2012; 10(10):717-25.-   2. Furet J P, Kong L C, Tap J, Poitou C, Basdevant A, Bouillot J L,    et al. Differential adaptation of human gut microbiota to bariatric    surgery-induced weight loss: links with metabolic and low-grade    inflammation markers. Diabetes. 2010; 59(12):3049-57.-   3. Monte S V, Caruana J A, Ghanim H, Sia C L, Korzeniewski K,    Schentag J J, et al. Reduction in endotoxemia, oxidative and    inflammatory stress, and insulin resistance after Roux-en-Y gastric    bypass surgery in patients with morbid obesity and type 2 diabetes    mellitus. Surgery. 2011.-   4. O'Mahony L, McCarthy J, Kelly P, Hurley G, Luo F, Chen K, et al.    Lactobacillus and bifidobacterium in irritable bowel syndrome:    symptom responses and relationship to cytokine profiles.    Gastroenterology. 2005; 128(3):541-51.-   5. Hajishengallis G, Chavakis T. Endogenous modulators of    inflammatory cell recruitment. Trends Immunol. 2012.-   6. Larsen N, Vogensen F K, van den Berg F W, Nielsen D S, Andreasen    A S, Pedersen B K, et al. Gut microbiota in human adults with type 2    diabetes differs from non-diabetic adults. PLoS One. 2010;    5(2):e9085.-   7. Vrieze A, Holleman F, Zoetendal E G, de Vos W M, Hoekstra J B,    Nieuwdorp M. The environment within: how gut microbiota may    influence metabolism and body composition. Diabetologia. 2010;    53(4):606-13.-   8. Maqbool S, Parkman H P, Friedenberg F K. Wireless capsule    motility: comparison of the SmartPill G I monitoring system with    scintigraphy for measuring whole gut transit. Dig Dis Sci. 2009;    54(10):2167-74.-   9. Gao X W, Mubasher M, Fang C Y, Reifer C, Miller L E.    Dose-response efficacy of a proprietary probiotic formula of    Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R for    antibiotic-associated diarrhea and Clostridium difficile-associated    diarrhea prophylaxis in adult patients. Am J Gastroenterol. 2010;    105(7): 1636-41.-   10. Johnson S, Maziade P J, McFarland L V, Trick W, Donskey C,    Currie B, et al. Is primary prevention of Clostridium difficile    infection possible with specific probiotics? Int J Infect Dis. 2012.-   11. Brenner D M, Moeller M J, Chey W D, Schoenfeld P S. The utility    of probiotics in the treatment of irritable bowel syndrome: a    systematic review. Am J Gastroenterol. 2009; 104(4):1033-49; quiz    50.-   12. Marik P E. Colonic flora, Probiotics, Obesity and Diabetes.    Front Endocrinol (Lausanne). 2012; 3:87.

1.-28. (canceled)
 29. A method of treating a gastrointestinal disorderin a subject in need thereof, the method comprising administering to thesubject a pharmaceutically effective amount of an oral formulationcomprising: a biodegradable first capsule comprising a probioticformulation targeted to the proximal colon, wherein the first capsulecomprises a reverse enteric coating that solubilizes in a pH of about6.2 to about 6.5; and a biodegradable second capsule that includes thefirst capsule and a probiotic formulation targeted to the ileum, whereinthe second capsule comprises an enteric coating that solubilizes in a pHof about 7 to 8, wherein upon administration to the subject, the secondcapsule releases the first capsule and the probiotic formulationtargeted to the ileum in the ileum and once released the first capsulereleases the probiotic formulation targeted to the proximal colon in theproximal colon.
 30. The method of to claim 29, wherein thegastrointestinal disorder is a Clostridium difficile disorder. 31.(canceled)
 32. The method of claim 30, wherein the Clostridium difficiledisorder is associated with one or more of a Clostridium difficileinfection, an imbalance of Clostridium difficile in the ileum or colonof the subject, diarrhea, inflammation, colitis, fever, or dysbiosis.33. The method of claim 29, wherein the probiotic formulation targetedto the proximal colon and the probiotic formulation targeted to theileum each comprise a live bacterial suspension comprising a bacteriaselected from the genus Lactobacillus and Bifidobacterium.
 34. Themethod of claim 29, wherein the gastrointestinal disorder is anirritable bowel disease associated with dysbiosis.
 35. (canceled) 36.The method of claim 29, wherein the oral formulation is administered incombination with a statin, linaclotide, an ileal brake hormone releasingsubstance, or an anti-inflammatory agent. 37.-45. (canceled)
 46. Themethod of claim 29, wherein the enteric coating comprises one or morepolymers each selected from the group consisting of copolymers ofmethacrylic acid, and copolymers of methacrylic acid and methylmethacrylate.
 47. The method of claim 29, wherein the biodegradablefirst capsule and the biodegradable second capsule each comprisehydroxypropylmethyl cellulose.
 48. The method of claim 29, wherein thebiodegradable first capsule and the biodegradable second capsule areeach band sealed.
 49. A method of treating a gastrointestinal disorderin a subject in need thereof, the method comprising administering to thesubject a pharmaceutically effective amount of a capsule-in-capsuleformulation comprising: an inner biodegradable capsule contained withinan outer biodegradable capsule, wherein the inner capsule contains aninner probiotics formulation and is coated with a reverse entericcoating that solubilizes in a pH of about 6.2 to about 6.5; and theouter capsule contains the inner capsule and an outer probioticsformulation, and is coated with an enteric coating that solubilizes in apH of about 7 to
 8. 50. The method of claim 49, wherein thegastrointestinal disorder is a Clostridium difficile disorder.
 51. Themethod of claim 49, wherein the gastrointestinal disorder is anirritable bowel disease associated with dysbiosis.
 52. The method ofclaim 49, wherein the reverse enteric coating comprisesdimethylaminoethyl methacrylate-butyl methacrylate-methyl methacrylatecopolymer (2:1:1).
 53. The method of claim 49, wherein the entericcoating comprises poly(methacrylic acid-co-methyl-methacrylate) 1:1copolymer and poly(methacrylic acid-co-methyl-methacrylate) 1:2copolymer.
 54. The method of claim 49, wherein the reverse entericcoating comprises 5 mg/cm² to 10 mg/cm² of dimethylaminoethylmethacrylate-butyl methacrylate-methyl methacrylate copolymer (2:1:1)copolymer and the enteric coating comprises 5 mg/cm² to 10 mg/cm² ofpoly(methacrylic acid-co-methyl-methacrylate) 1:1 copolymer andpoly(methacrylic acid-co-methyl-methacrylate) 1:2 copolymer in a 75/25ratio.
 55. The method of claim 49, wherein the reverse enteric coatingand the enteric coating each have a thickness of 60 μm to 180 μm. 56.The method of claim 49, wherein the inner capsule and outer capsule eachcomprise hydroxypropylmethyl cellulose.
 57. The method of claim 49,wherein the inner capsule is a size no. 3 hydroxypropylmethyl cellulosecapsule.
 58. The method of claim 49, wherein the outer capsule is a sizeno. 0 hydroxypropylmethyl cellulose capsule.
 59. The method of claim 49,wherein the inner capsule and outer capsule each are each band sealed.